R&D Systems Products for Neuroscience Research

R&D Systems Tools for Cell Biology Research™

R&D Systems Products for Neuroscience Research ON THE COVER

This illustration was featured in the Cytokine Bulletin (2010, Issue 1).

TLR2 IL-6 IL-6 R MOG IL-21 R

TGF-β R STAT3 Dendritic TGF-β Cell STAT3 Batf, RORγt IL-23 Batf RORγt Th17 Cell IL-23 R IL-17 JunB Batf RORγt

IL-17 NO

Myelin TNF-α Axon MMPs

Macrophages Neuron Oligodendrocyte

Prolonged production of IL-17 by Th17 cells is dependent on the transcription factor Batf. Recent studies suggest that prolonged production of IL-17 by Th17 cells is dependent on the synergistic actions of the RORt and Batf-JunB transcription factors. These fi ndings may have implications for Th17-related autoimmune disorders such as multiple sclerosis. Following induction of autoimmune conditions in mice using myelin oligodendrocyte glycoprotein (MOG) immunization, diff erentiated Th17 cells secrete proinfl ammatory cytokines, and activated macrophages destroy myelin and damage oligodendrocytes. It remains to be determined whether the induction of Batf expression is dependent on STAT3 in Th17 cells, and whether an interaction between Batf and Irf4 or Ahr is required to promote the respective induction of IL-21 and IL-22. Schraml, B.U. et al. (2009) Nature 460:405.

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R&D Systems Tools for Cell Biology Research™

R&D Systems Cytokine Bulletin Cytokine BULLETIN 2011 | Issue 2

Adipose Tissue INSIDE s)NFLAMMATION s$ECREASEDADIPOGENESIS IL-1E PAGE 2 s2EDUCEDADIPOCYTEMETABOLICACTIVITY Synaptic EphB2-NMDA Receptor s)NSULIN2ESISTANCE Dynamics: A New Hope for Alzheimer’s Disease Obesity-related Adipocyte Danger Signal PAGE 3 CXCR4: A Receptor Macrophage ROS for Extracellular Ubiquitin Pro-IL-1E PAGE 4 Nlrp3 TSLP, Inflammation, & Cancer PAGE 5 Pancreas Caspase-1 Mature IL-1E RECENT CITATIONS: R&D Systems Products for TGF-E ASC Research IsletIslet ofof LangerhansLangerhans Pro-Caspase-1 PAGE 6-7 TECHNICAL NOTE & MEETINGS: something PAGE 8 IL-1E s)MPAIREDPANCREATICE cell function NEW TOOLS: sE cell death Custom Premix Fluorokine® Multianalyte Profiling Kits Obesity-Induced Inflammasome Activation in Key Metabolic Tissues Promotes Chronic Inflammation and Insulin Resistance. Obesity-related danger signals, such as palmitate, & VersaMAP™ Development System ceramide, high glucose concentrations, islet amyloid polypeptide, defective autophagy, or mitochondrial dysfunction may lead to the generation of reactive oxygen species (ROS) and subsequent activation of the Nlrp3 inflammasome in adipocytes, pancreatic islet cells, or infiltrating macrophages present in these tissues. Nlrp3 inflammasome activation promotes the cleavage and activation of Caspase-1, resulting in the secretion of IL-1E, a proinflammatory cytokine that negatively affects adipocyte differentiation, inhibits insulin signaling, and has BIObrief Mini Poster a cytotoxic effect on insulin-producing pancreatic E cells. Identification of a connection between obesity, the inflammasome, and insulin resistance links inflammasome activation hwit The Double Life of TGF-E: metabolic tissue dysfunction, which may contribute to the pathogenesis of type II diabetes, and potentially other diseases associated with obesity-induced inflammation. A Tumor Suppressing & Tumor Obesity-induced Activation of the Nlrp3 Inflammasome Promoting Factor Promotes Insulin Resistance Obesity is a serious health problem characterized by an excessive expansion of the white adipose tissue coupled with a state of chronic, low-grade inflammation.1 Inflammation occurs as a result of immune cell infiltration of the adipose tissue and increased production of pro-inflammatory cytokines.2 These changes www. RnDSystems.com negatively affect normal adipocyte functions, such as triglyceride storage and lipolysis, leading to high circulating levels of free fatty acids and ectopic lipid accumulation.3 In addition, elevated levels of pro- inflammatory cytokines, including IL-1E, IL-6, and TNF-D, reduce insulin sensitivity, which can lead to the development of type II diabetes if insulin production by the pancreatic E cells is not sufficiently increased.4-6 Although it has been recognized for some time that pro-inflammatory factors inhibit insulin signaling, the molecular mechanisms that trigger inflammation and sustained cytokine production during obesity are not well understood. The first evidence of an obesity-related signal that could initiate the inflammatory response came from reports that saturated fatty acids activate Toll-like receptors (TLRs).7-9 TLRs are a family of membrane- associated pattern recognition receptors that detect invading pathogens and activate the innate immune response. These studies demonstrated that fatty acids, like microbial pathogens, promote TLR-induced inflammation. It was subsequently shown that disruption of TLR4 or a loss of function mutation in TLR4 protects against high fat diet-induced insulin resistance in mice, suggesting that fatty acid-induced TLR4 signaling provides a link between obesity, inflammation, and insulin resistance.10,11 Continued on page 7 TABLE OF CONTENTS

R&D SYSTEMS NEUROSCIENCE CATALOG The fi eld of neuroscience is one of the most fascinating and complex branches of biological science. At the earliest stages of development, the regulated expression and activity of specifi c genes directs multipotent stem cells down neural pathways. Morphogenic factors regulate neuroectoderm diff erentiation, the formation of the neural tube, and fi nally the development of the brain and spinal cord. The growth cones at the tips of extending axons are exquisitely sensitive to a myriad of environmental cues that govern their directed migration toward specifi c targets. Once there, the process of synaptogenesis incorporates neurons into complex electrochemical networks. By adulthood, the brain may contain more than 100 trillion synapses, their integrated signals controlling virtually all aspects of life. With age, the nervous system becomes more vulnerable to dysfunction and disease. For instance, the risk of Alzheimer's disease doubles every 5 years after the age of 65 and reaches 50% by the age of 85. We advance our understanding of nervous system pathology with the hope of developing therapies to address some of society's most economically and socially devastating diseases. R&D Systems off ers a range of high quality reagents for studying all areas of neuroscience, from neural stem cells to neurodegenerative disease. These include a wide range of high performance antibodies, and the most referenced collection of bioactive proteins and immunoassays in the industry. Primary rat and mouse cortical stem cells are also available, as well as kits for the expansion, diff erentiation, and identifi cation of neural stem cells, and multiplex assays for the simultaneous detection of multiple analytes.

NEUROSCIENCE-RELATED MOLECULE FAMILIES

NEUROTROPHIC FACTORS & RECEPTORS ...... 2-9 NEURAL STEM CELLS ...... 36-43 GDNF Family Ligands & Receptors ...... 2-3 Neural Stem Cells & Diff erentiation Markers ...... 36-41 Neurotrophin Family Ligands & Receptors ...... 4-5 Adult Neurogenesis ...... 42-43

VPS10P-domain Receptor Family ...... 6-7 SYNAPTIC PROTEINS & NEUROTRANSMITTERS ...... 44-49 Additional Neurotrophic Factors & Receptors ...... 8-9 Synaptic Proteins ...... 44-47 NEURAL DEVELOPMENT & AXON GUIDANCE ...... 10-29 Neurotransmitters, Receptors, & Related Molecules ...... 48-49

Ephrin Ligands & Eph Receptors ...... 10-11 NEURODEGENERATIVE DISEASES ...... 50-86 Hedgehog Family ...... 12-13 Alzheimer’s Disease & APP Metabolism ...... 50-51 Netrin Family ...... 14-15 Parkinson’s Disease ...... 52-53 Semaphorins, Plexins, & Related Molecules ...... 16-17 Amyotrophic Lateral Sclerosis ...... 54-55 Slit Ligands & ROBO Receptors ...... 18-19 Multiple Sclerosis ...... 56-57 Wnt Ligands, Frizzled Receptors, & Related Molecules ...... 20-23 Diabetic Peripheral Neuropathy ...... 58-59 Nogo Proteins & Receptors ...... 24-25 Neuroinfl ammation ...... 60-61 Bone Morphogenetic Proteins & Receptors ...... 26-27 Autophagy & the Ubiquitin Proteasome System ...... 62-65 Repulsive Guidance Molecules & Receptors ...... 28-29 Apoptosis ...... 66-73 Additional Axon Guidance Molecules ...... 29 SIGNAL TRANSDUCTION ...... 74-86 CELL ADHESION MOLECULES FOR NEUROSCIENCE ...... 30-35 SPECIALIZED TOOLS FOR NEUROSCIENCE RESEARCH ...... 87-105 Contactin Family...... 30-31 Antibody Arrays & Multiplex Assays ...... 87-94 Nectins & Cadherins...... 32-33 Neural Stem Cell Culture Media & Kits ...... 94-97 SynCAM, NCAM, & L1CAM Families ...... 34-35 Neuronal Cell Culture Products ...... 97-98 Flow Cytometry, Western Blotting, & Cell/Tissue Staining ...... 99-104 Chromatin IP Kits & Modules ...... 105

NOTES ...... 106-108

For more information visit our website at www.RnDSystems.com/go/Neuroscience NEUROTROPHIC FACTORS & RECEPTORS

GDNF Family Ligands & Receptors Following its original purifi cation from a rat glioma cell line supernatant, Although GFR  is linked to the plasma membrane at lipid rafts by a glial cell line-derived neurotrophic factor (GDNF) was identifi ed as a glycosylphosphatidylinositol (GPI) anchor, cleavage by proteases or survival factor for dopaminergic neurons in the midbrain.1 Subsequent phospholipases generates a soluble form of the receptor that can exert studies have shown that GDNF is critical for the proliferation, migration, distant actions. 2,3 and diff erentiation of several neuronal populations. In addition, GDNF Many groups have studied GDNF as a potential therapeutic agent for the exerts important biological eff ects outside the central nervous system, treatment of Parkinson’s disease (PD), a condition that is characterized including promoting urinary collecting duct formation in the kidneys and by loss of dopaminergic neurons in the pars compacta of the substantia 4,5 spermatogonial diff erentiation in the testes. nigra. Studies have shown that GDNF has neuroprotective and restorative The GDNF family of ligands (GFL) includes the other cysteine-knot proteins, eff ects when administered against a variety of neurotoxins both in vitro Neurturin, Artemin, and Persephin. All GFLs are produced as prepro-GFL and in animal models of PD.10,11 However, the clinical eff ects of GDNF have precursors that are activated following proteolytic cleavage. Although been disappointing, possibly because its heparin-binding domains and some cross-talk exists, GFLs preferentially signal as homodimers through large molecular size hinder delivery of physiologically relevant levels in their cognate GDNF-family receptor  subtype (GFR , Figure 1). All GFR  the brain. Thus, developing a viable method of delivery to the midbrain is subtypes are concentrated in areas of the plasma membrane called lipid a focus of PD-related research. rafts, specialized microdomains that facilitate the colocalization of proteins References involved in intracellular signaling pathways. Following ligand binding, 1. Lin, L.F. et al. (1993) Science 260:1130. GFL-GFR  complexes associate with the extracellular domain of the c-Ret receptor tyrosine kinase, which activates by auto phosphorylation and 2. Airaksinen, M.S. & M. Saarma (2002) Nat. Rev. Neurosci. 3:383. induces a postulated common signaling pathway (Figure 2). Studies have 3. Paratcha, G. & F. Ledda (2008) Trends Neurosci. 31:384. shown that GDNF binding to GFR -1 recruits c-Ret to lipid rafts, an action 4. Tee, J.B. et al. (2010) Dev. Biol. 347:337. that is dependent on TGF- .6  5. Meng, X. et al. (2000) Science 287:1489.

GDNF can also signal independently of c-Ret through interactions be- 6. Peterziel, H. et al. (2002) J. Biol. Chem. 159:157. tween GFR -1 and Neuronal Cell Adhesion Molecule 1 (NCAM-1), which 7. Ibáñez, C.F. et al. (2010) Biochem. Biophys. Res. Commun. 396:24. activates Fyn and Focal Adhesion Kinase (FAK). 7 In addition, through its 8. Sjöstrand, D. & C.F. Ibáñez (2008) J. Biol. Chem. 283:13792. association with NCAM, GDNF-GFR -1 can regulate homo philic cell-cell adhesion.8 Recent studies revealed that GDNF, Neurturin, and Artemin 9. Bespalov, M.M. et al. (2011) J. Cell Biol. 192:153. also induce neurite outgrowth via interactions with Syndecan-3, a 10. Kells, A.P. et al. (2010) J. Neurosci. 30:9567. 9 transmembrane heparan sulfate proteoglycan, and Src kinase activation. 11. Boscia, F. et al. (2009) PLoS One 4:e6486.

GDNF Neurturin Artemin Persephin Neurturin GDNF GDNF Neurturin NCAM-1 Artemin Artemin c-Ret

GFR D-1 c-Ret GFR D-1

sGFR D-1

Extracellular

Plasma Membrane GFR D-3 GFR D-4 Fyn GFR D-2 Intracellular GFR D-1 SHC PLC-J SHC FAK PLCJ Ras/MAPK PI 3-K/Akt PLC-J/Ca2+ Cdk5

Domain Key: GDNF-family ligand homodimer Globular GPI anchor Cholesterol Glycosphingolipid Cadherin-like Cysteine-rich Tyrosine kinase Phosphorylation site Ig-like Leucine-rich

Figure 1. GDNF Family Ligands and Receptors. GDNF family ligands signal as homodimers through their cognate GDNF family Figure 2. GDNF Receptor Signaling Pathways. All GFR -ligand complexes associate with Ret to induce autophosphorylation, receptor  (GFR ), with some cross-talk. Major ligand receptor interactions are shown in color. Minor interactions are shown dimerization, and signal transduction via the indicated signaling pathways (dimerization not shown for illustrative purposes). In in black text. At the plasma membrane, all GFR  members are concentrated in lipid rafts, specialized microdomains that are addition, GDNF-GFR -1 interacts with Neuronal Cell Adhesion Molecule 1 (NCAM-1) to transduce signals through the Fyn/Focal enriched with cholesterol, glycoshingolipids, and glycosylphosphatidylinositol (GPI)-anchored proteins. Adhesion Kinase (FAK) pathway. Following cleavage by membrane-associated phospholipases, soluble GFR -1 (sGFR -1) is shed from the cell surface and can induce non-cell-autonomous actions through Ret/Cdk5.

2 For research use only. Not for use in diagnostic procedures. NEUROTROPHIC FACTORS & RECEPTORS

R&D Systems Products for GDNF Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

GDNF Family Ligands IRS2 H (WB)

Artemin H M H (IHC, WB) M (B/N, IHC, WB) Jak1 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB)

GDNF H R H (B/N, ELISA, IHC, WB) R (B/N, IHC, WB) H Jak2 M (WB) R (WB)

Neurturin H M H (B/N, IHC, WB) M (B/N, IHC, WB) Jak3 H (FC, WB)

Persephin H M H (IHC, WB) M (WB) JNK H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R

GDNF Family Receptors JNK1/JNK2 H (IHC, WB) M (IHC, WB) R (IHC, WB)

Gas1 H M H (ELISA, WB) M (ELISA, IHC, WB) H M JNK1 MH (IHC, WB) M (IHC, WB) R (IHC, WB)

GFR-1/GDNF R-1 H R H (IHC, WB) R (B/N, IHC, WB) JNK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R

GFR-2/GDNF R-2 H M H (B/N, IHC, WB) M (B/N, IHC, WB) p38 MAP Kinase H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M

GFR-3/GDNF R-3 H M H (IHC, WB) M (IHC, WB) p38 HH (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M R

GFR-4/GDNF R-4 H (WB) M (IHC, WB) p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M

GFR-like M (IHC, WB) p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M R

NCAM-1/CD56 H M H (ELISA, FC, IHC, WB) M (WB) R (WB) H p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M

Ret H M H (FC, IHC, WB) M (IHC, WB) H PDK-1 HH (WB) M (WB) R (WB)

Syndecan-3 H M H (FC, IHC, WB) M (IHC, WB) PI 3-Kinase p55 H (WB) M (WB) R (WB)

GDNF Family Signaling Molecules PI 3-Kinase p85 H (WB) M (WB) R (WB)

Akt H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R PI 3-Kinase p110 H (WB)

Akt1 HH (FC, IHC, WB) M (WB) R (WB) H M R PI 3-Kinase p110 H (WB)

Akt2 H (FC, IHC, WB) M (IHC, WB) R (IHC, WB) H PI 3-Kinase p110 H (WB)

Akt3 H (FC, WB) PLC-1 H (WB) M (WB)

CREB H (ChIP, IHC, WB) M (WB) R (WB) H M R PLC-3 H (WB)

DOK4 H (IHC, WB) PLC-4 H (WB) M (WB) R (WB)

ERK1/ERK2 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R PLC-1 H (WB) M (WB) R (WB)

ERK1 HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R PLC-2 H (IHC, WB) M (IHC, WB)

ERK2 HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R PLC-3 H (WB) M (WB)

ERK3 H (IHC, WB) Ras H (WB) M (WB) R (WB)

FAK HH (IHC, WB) M (IHC, WB) R (IHC, WB) H M R Ret H M H (FC, IHC, WB) M (IHC, WB)

FRS2 H (FC, IHC, WB) M (WB) R (WB) H M R Src H V H (IHC, WB) M (IHC, WB) R (IHC, WB) H

Gab2 H (WB) STAT3 H (ChIP, FC, IHC, IP, WB) M (ChIP, FC, IHC, IP, WB) H M R (ChIP, FC, IHC, IP, WB) GRB7 H (WB) M (WB) R (WB) Tyk2 H (WB) IRS1 H (IHC, WB) M (WB) R (WB) Phospho-Tyrosine Ms (FC, IHC, IP, WB) Species Key: H Human M Mouse R Rat Ms Multispecies V Viral Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FC Flow Cytometry WB Western blot

Neurturin Antibody (Pg/mL) 10-2 10-1 100 101 102 0.8 0.8 Cell Survival (Mean O.D.)Cell Survival

0.7 0.7

0.6 0.6

0.5 0.5 Cell SurvivalCell (Mean O.D.)

0.4 0.4 10-1 100 101 102 Neurturin (ng/mL)

Detection of GFR -like in Mouse Brain. GDNF Receptor (GFR) -like was detected Neuron Survival in Response to Neurturin and Neutralization by a Neurturin GDNF and GFR-1 in Rat Dorsal Root Ganglia. Glial Cell Line-derived Neurotrophic in immersion-fi xed frozen sections of mouse brain using a Sheep Anti-Mouse GFR Antibody. Recombinant Human Neurturin (Catalog # 1297-NE-025) supports Factor (GDNF) and GDNF Receptor -1 (GFR-1) were detected in perfusion-fi xed -like Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5728). The tissue the survival of dorsal root gangli on neurons from E11 chick embryos in a dose- frozen sections of rat dorsal root ganglia using a Goat Anti-Human/Rat GDNF was stained using the NorthernLights™ 557-conjugated Donkey Anti-Sheep IgG dependent manner (purple line), as measured by MTT. Neuronal survival elicited by Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF-212-NA; green) and a Secondary Antibody (Catalog # NL010; red) and counterstained (green). Specifi c Recombinant Human Neurturin (6 ng/mL) is neutralized (green line) by increasing Biotinylated Goat Anti-Rat GFR -1 Antigen Affi nity-purifi ed Polyclonal Antibody staining was localized to medullary gigantocellular neurons in the brainstem. concentrations of a Goat Anti-Human Neurturin Antigen Affi nity-purifi ed Polyclonal (Catalog # BAF560; red). Antibody (Catalog # AF387). www.RnDSystems.com/go/GDNF 3 NEUROTROPHIC FACTORS & RECEPTORS

Neurotrophin Family Ligands & Receptors The neurotrophin family includes the structurally related growth factors NGF R is a member of the tumor necrosis factor (TNF) receptor superfamily -Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), of death receptors and shares overall structure with TNF- p55 receptor Neurotrophin-3 (NT-3), and Neurotrophin-4 (NT-4). These secreted (although it cannot bind TNF-). Unlike Trk receptors, all neurotrophins proteins infl uence neurite outgrowth, synaptogenesis, synaptic plasticity, bind to NGF R with equal affi nity but diff erent kinetics. Recent reports neuronal survival and diff erentiation, and myelination.1 All neurotrophins indicate that in the absence of neurotrophins, NGF R exists as a dimer are generated as 30-35 kDa proteins containing prepro- and pro-domains, that is disulphide-linked through a highly conserved Cys257 in the trans- which are proteolytically cleaved to yield the secreted 12-13 kDa mature membrane domain.7 Latest research suggests that neurotrophin-induced proteins. Members of the cysteine knot family, neurotrophins function as NGF R-dependent apoptosis requires activation of c-Jun terminal kinase non-covalently associated symmetrical homodimers. (JNK), receptor-induced proteolysis via upregulated TNF- converting Pro-neurotrophins are also known to be biologically active and often enzyme (TACE) and -Secretase, and nuclear translocation of ubiquitinated neurotrophin receptor-interacting factor (NRIF) (Figure 4).8 Other groups exert actions that are distinct from the mature form.2 Alternatively, pro- neurotrophins can be secreted and subsequently cleaved by extracellular have demonstrated that in the presence of the co-receptor Sortilin, pro- 9 proteases, including Matrix Metalloproteinase-3 (MMP-3), MMP-9, NGF and pro-BDNF bind NGF R and induce apoptosis. Finally, recent studies support the hypothesis that TrkA and TrkC are dependence receptors that and Plasmin.3 There are two distinct types of neurotrophin receptors, induce apoptosis in the absence of their respective ligands.10 tropomyosin kinase (Trk) receptors and neurotrophin receptor p75 (NGF R). Trophic actions that promote neuronal survival are dependent on References mature neurotrophins binding to Trk receptors. In contrast, apoptosis is 1. Caparali, A. & C. Emanueli (2009) Physiol. Rev. 89:279. transduced by mature and pro-domain neurotrophins through NGF R. 2. Lee, R. et al. (2001) Science 294:1945.

Trk receptors are single-pass transmembrane proteins that contain highly 3. Mowla, S.J. et al. (2001) J. Biol. Chem. 276:12660. conserved intracellular, tyrosine kinase, and enzymatically active domains. 4. Clary, D.O. & Reichardt, L.F. (1994) Proc. Natl. Acad. Sci. USA 91:11133. Ligand binding occurs in the more variable immunoglobulin (Ig)-like C2 domains (Figure 3).4,5 Trk receptors dimerize following ligand binding. 5. Strohmaier, C. et al. (1996) EMBO J. 15:3332. Phosphorylation of tyrosine residues within the intracellular domain 6. Mischel, P.S. et al. (2001) J. Biol. Chem. 276:11294. promotes association of the receptor with adaptor proteins and signaling 7. Vilar, M. et al. (2009) Neuron 62:72. molecules, that mediate a myriad of physiological responses. Studies have 8. Kenchappa, R.S. et al. (2010) J. Biol. Chem. 285:20359. shown that NGF R will associate with all three Trk receptors to promote 9. Willnow, T.E. et al. (2008) Nat. Rev. Neurosci. 9:899. high-affi nity binding and increase ligand discrimination.6 10. Nikoletopoulou, V. et al. (2010) Nature 467:59.

Neurotrophin Family Neuronal Survival Apoptosis E-NGF BDNF NT-4 NT-3 Pro-NTs & Mature NTs

Trk Trk NGF RECD

NGF R NGF R

Extracellular Sortilin NGF R Plasma Membrane TACE J-Secretase NRAGE Intracellular NGF R NGF R SHC NRIF ICD TrkC PLC-J TrkB TRAF6 JNK3 JNK3 TrkA NFNB NRIFUb 2+ JNK Ras/MAPK/ERK PLC-J/Ca TACE PI 3-K/Akt JNK3 Apoptosis Nucleus

Ub Domain Key: Neurotrophic dimer Pro-neurotrophin Cysteine-rich Leucine-rich Ig-like Tyrosine kinase Phosphorylation site Death domain VPS10P Fibronectin type III Ubiquitination

Figure 3. Neurotrophin Family Ligands and Receptors. Mature neurotrophins function as symmetrical homodimers by binding Figure 4. Neurotrophin Signaling Exerts Paradoxical Eﬀ ects on Cell Viability. During development, the balance of Trk tropomyosin kinase (Trk) receptors and neurotrophin receptor p75 (NGF R). Ligand binding to Trk receptors occurs within the receptors, NGF R, mature neurotrophins, and pro-neurotrophins determines cell fate. Neuronal survival is transduced by Trk two immunoglobulin (Ig)-like domains. In addition to the established cognate relationships, NT-3 has been shown to bind TrkA receptors alone, or in association with NGF R. Apoptosis is induced by mature neurotrophins via NGR R. This process is believed and TrkB. Mature neurotrophins and neurotrophins containing a pro-domain bind NGF R within the four cysteine-rich domains. to require activation of c-Jun terminal kinase (JNK), receptor-induced proteolysis via upregulated TNF- converting enzyme (TACE), further cleavage by -Secretase, and nuclear translocation of neurotrophin receptor-interacting factor (NRIF) following TNF receptor-associated factor 6 (TRAF6)-dependent ubiquitination. Pro-neurotrophins also induce apoptosis by binding to NGF R associated with Sortilin. 4 For research use only. Not for use in diagnostic procedures. NEUROTROPHIC FACTORS & RECEPTORS

R&D Systems Products for Neurotrophin Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Neurotrophin Family Ligands NFB1 H (ChIP, WB) M (ChIP, WB) H

BDNF HH (ELISA, FC, IHC, WB) H NFB2 H (ChIP, IHC, WB) M (WB) H

-NGF H M R H (B/N, ELISA, IHC, WB) R (B/N, ELISA, IHC, WB) H R NRAGE H (FC, IHC, WB) M (WB) R (WB)

NT-3 HH (B/N, ELISA, IHC, WB) H p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M

NT-4 H M H (B/N, ELISA, IHC, WB) H p38 HH (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M R

Neurotrophin Family Receptors p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M

NGF R/TNFRSF16 H M H (FC, IHC, WB) M (IHC, WB) p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M R

Sortilin H M H (B/N, IHC, WB) M (B/N, IHC, WB) p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M

TrkA H R H (B/N, FC, IHC, WB) M (IHC, WB) R (IHC, WB) H R PDK-1 HH (WB) M (WB) R (WB)

TrkB H M H (FC, IHC, WB) M (B/N, IHC, WB) H PI 3-Kinase p110 H (WB)

TrkC H M H (B/N, FC, IHC, WB) M (B/N, IHC, WB) H PI 3-Kinase p110 H (WB)

TROY/TNFRSF19 H M H (WB) M (ELISA, IHC, WB) M PI 3-Kinase p110 H (WB)

Neurotrophin Family Signaling Molecules PI 3-Kinase p55 H (WB) M (WB) R (WB)

Akt H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R PI 3-Kinase p85 H (WB) M (WB) R (WB)

Akt1 HH (FC, IHC, WB) M (WB) R (WB) H M R PLC-1 H (WB) M (WB)

Akt2 H (FC, IHC, WB) M (IHC, WB) R (IHC, WB) H PLC-3 H (WB)

Akt3 H (FC, WB) PLC-4 H (WB) M (WB) R (WB)

ERK1 HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R PLC-1 H (WB) M (WB) R (WB)

ERK1/ERK2 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R PLC-2 H (IHC, WB) M (IHC, WB)

ERK2 HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R PLC-3 H (WB) M (WB)

ERK3 H (IHC, WB) Ras H (WB) M (WB) R (WB)

ERK4 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ras-GAP H (WB) M (WB) R (WB)

ERK5/BMK1 H (IHC, WB) M (IHC, WB) TACE/ADAM17 H M H (ELISA, FC, IHC, IP, WB) H

JNK1 MH (IHC, WB) M (IHC, WB) R (IHC, WB) TRAF-6 H (WB)

JNK1/JNK2 H (IHC, WB) M (IHC, WB) R (IHC, WB)

JNK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R

Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot

p-TrkA p-TrkB p-TrkC A. B. 50 Untreated Untreated Untreated ) 3 40 E-NGF BDNF NT-3

Untreated 30 Treated

10 Mean Pixel Intensity (x10 Intensity Mean Pixel

0 Phospho-TrkA Phospho-TrkB Phospho-TrkC

TrkB in Mouse Spinal Cord. TrkB was detected in perfusion-fi xed frozen sections of Detection of Neurotrophin-induced Receptor Tyrosine Kinase Phosp horylation -NGF Enhances Neurite Outgrowth. Chick dorsal root ganglion neurons (E10-11) mouse spinal cord using a Goat Anti-Mouse TrkB Antigen Affi nity-purifi ed Polyclonal using the Proteome Proﬁ ler™ Human Phospho-RTK Array. The C6 rat glial cell were cultured for 3-days under control conditions (A) or in the presence of Antibody (Catalog # AF1494). The tissue was stained using the NorthernLights™ line was transfected with human TrkA, TrkB, or TrkC. Cells were untreated, or treated 16 ng/mL Recombinant Human -NGF (Catalog # 256-GF; B). The presence of -NGF 557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001; red) and with Recombinant Human -NGF (Catalog # 256-GF), Recombinant Human BDNF signifi cantly enhanced neurite outgrowth. counterstained with DAPI (blue). Specifi c staining was localized to neuronal processes (Catalog # 248-BD), or Recombinant Human NT-3 (Catalog # 267-N3) for 5 minutes. and cell bodies. Cell lysates were assessed for the relative levels of phosphorylation of TrkA, TrkB, TrkC and nine other receptor tyrosine kinases (RTKs) using the Proteome Profi ler Human Phospho-RTK Array Kit (Catalog # ARY001). Representative images of the pre-spotted nitrocellulose membranes are shown following chemiluminescent detection (inset).

www.RnDSystems.com/go/Neurotrophins 5 NEUROTROPHIC FACTORS & RECEPTORS

VPS10P-domain Receptor Family Vacuolar protein sorting 10 protein (VPS10P)-domain receptors are type Dysregulated protein traffi cking by VPS10P-domain receptors has been 1 transmembrane proteins that bind a wide range of ligands including implicated in various disease conditions including Alzheimer’s disease neurotrophins, neuropeptides, and other transmembrane proteins.1 (AD) and other dementias, type-2 diabetes, and coronary artery disease. There are fi ve vertebrate members of the family, Sortilin, sorting protein- A pathological hallmark of AD is the increased generation of amyloid- related receptor with A-type repeats (SorLA), sortilin-related receptor CNS protein (A) and its deposition in the brain as senile plaques. Many expressed 1 (SorCS1), SorCS2, and SorCS3 (Figure 5). These multifunctional groups have reported an inverse correlation between the expression molecules have been shown to aff ect neuronal viability and function by levels of SorLA and A in vivo and in vitro.9,10,11 Recent studies suggest the regulating protein transport and signal transduction. Each receptor is underlying mechanism may be dependent on loss of SorLA-dependent expressed in distinct neuronal populations, suggesting discrete functions traffi cking of Amyloid Precursor Protein (APP).12,13 In the presence of in diff erent cell types. SorLA, the majority of APP is confi ned to the TGN, and internalized APP Variable function between family members is also supported by the from the plasma membrane undergoes retrograde traffi cking from the distinct subcellular expression profi les of each receptor. Sortilin and SorLA early endosome back to the TGN (Figure 6A). In the absence of SorLA, are pre dominantly found intracellularly, in the trans-Golgi network (TGN), increased traffi cking to the plasma membrane and internalization of APP facilitates - and -Secretase cleavage and greater A production in with less than 10% at the cell surface.2 Subcellular traffi cking of SorCS1 is    endocytic compartments (Figure 6B). dependent on the splice variant. SorCS1a is predominantly intracellular, SorCS1b is primarily expressed at the cell surface, and SorCS1c is evenly References 3,4 divided between the two. SorCS2 and SorCS3 are also cell surface 1. Willnow, T.E. et al. (2008) Nat. Rev. Neurosci. 9:899. receptors, but do not appear to direct major intracellular traffi cking.5 In 2. Nielsen, M.S. et al. (2001) EMBO J. 20:2180. all family members, the VPS10P domain is believed to adopt a -propeller 3. Nielson, M.S. et al. (2008) Traffi c 9:980. fold structure and functions as the protein interaction motif.6 4. Hermey, G. et al. (2003) J. Biol. Chem. 278:7390. Neuroscience-related research of VPS10P-domain receptors has focused on two main areas, modulation of neurotrophin (NT) signaling and amyloid 5. Westergaard, U.B. et al. (2005) FEBS Lett. 579:1172. precursor protein (APP) cleavage. The neurotrophin family includes -NGF, 6. Paiardini, A. & V. Caputo (2008) Neuropeptides 42:205. BDNF, NT-3, NT-4. In addition, pro-neurotrophin precursors are known to 7. Nykjaer, A. et al. (2004) Nature 427:843. be secreted functional molecules that induce signaling pathways distinct 8. Bronfman, F.C. (2007) J. Neurochem. 109:91. from the corresponding cleaved mature forms. In general, mature NTs 9. Lane, R.F. et al. (2010) J. Neurosci. 30:13110. exert their biological eff ects on neuronal viability through tropomyosin kinase (Trk) receptors. Trk signaling is known to be potentiated by 10. Dodson, S.E. et al. (2010) J. Neurosci. 28:12877. Trk-dependent cleavage of the intracellular domain of neurotrophin 11. Rohe, M. et al. (2010) J. Neurosci. 29:15472. receptor p75 (NGF R). In contrast, pro-neurotrophins induce apoptosis in 12. Schmidt, V. et al. (2007) J. Biol. Chem. 282:32956. neurons via NGF R, an action that is not dependent on Trk receptors but is 13. Viera, S.I. et al. (2010) Mol. Neurodegen. 5:40. dependent on interactions between both the pro-neurotrophin domain and Sortilin, and the mature domain and NGF R.7 In addition to aff ecting ligand binding, Sortillin may also modulate neurotrophin signaling by A. B. regulating intramembrane proteolysis of NGF R.8 APP sAPPD APP

D-Secretase CTFD

Early Endosome E D SorCS3 Sortilin J SorCS1 SorCS2 NGF R E-Secretase SorLA APP J-Secretase Late Endosome sAPPE APP E-Secretase J-Secretase AE Trans-Golgi Network AICD Domain Key: VPS10P EGF repeat Complement-type repeat Fibronectin type III Leucine-rich Cysteine-rich Death domain SorLA

Figure 5. VPS10P-domain Receptor Family Structure and Interactions. Members of the mammalian VPS10P-domain receptor Figure 6. SorLA Modulates APP Traﬃ cking and A Production. A. Under normal physiological conditions SorLA binds APP and family include sorting protein-related receptor with A-type repeats (SorLA), sortilin-related receptor CNS expressed 1 (SorCS1), confi nes it to the trans-Golgi network (TGN). At the plasma membrane -Secretase cleaves APP to generate soluble APP (sAPP) SorCS2, and SorCS3, and Sortilin. Also shown are amyloid precursor protein (APP) and neurotrophin receptor p75 (NGF R). The and C-terminal fragment (CTF). In addition, SorLA sorts internalized APP for retrograde traffi cking from the early endosome back functions of APP and NGF R are modulated by SorLA and Sortilin, respectively. to the TGN. B. In the absence of SorLA, more APP is expressed at the plasma membrane and subsequently internalized to late endosomes, where it is cleaved by - and -Secretase to generate soluble APP (sAPP), A, and APP intracellular domain (AICD).

6 For research use only. Not for use in diagnostic procedures. NEUROTROPHIC FACTORS & RECEPTORS

R&D Systems Products for VPS10P-domain Repceptor Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

VPS10P-domain Receptors CNTF R H R H (B/N, WB) R (B/N, IHC, WB)

SorCS1 H M H (IHC, WB) Glut4 R (AP, IHC, IP, WB)

SorCS2 H M H (WB) M (IHC, WB) gp130 H M R H (B/N, ELISA, FC, WB) M (B/N, ELISA, FC, IHC, WB) H M

SorCS3 H M H (WB) M (B/N, IHC, WB) NGF R/TNFRSF16 H M H (FC, IHC, WB) M (IHC, WB)

SorLA H (IHC, WB) M (IHC, WB) Nicastrin H (IHC, WB)

Sortilin H M H (B/N, IHC, WB) M (B/N, IHC, WB) NT-3 HH (B/N, ELISA, IHC, WB) H

VPS10P-domain Related Molecules NT-4 H M H (B/N, ELISA, IHC, WB) H

APP/Protease Nexin II HH (IHC, IP, WB) M (IHC, WB) R (WB) Ca (WB) Ch (WB) H M R Progranulin/PGRN H M H (IHC, IP, WB) M (ELISA, IHC, WB) H M Pr (WB) Ms (WB) TrkA H R H (B/N, FC, IHC, WB) M (IHC, WB) R (IHC, WB) H R BACE-1 H M H (B/N, FC, IHC, IP, WB) M (B/N, FC, IHC, IP, WB) TrkB H M H (FC, IHC, WB) M (B/N, IHC, WB) H BACE-2 MH (IHC, IP, WB) M (IP, WB) TrkC H M H (B/N, FC, IHC, WB) M (B/N, IHC, WB) H BDNF HH (ELISA, FC, IHC, WB) H TROY/TNFRSF19 H M H (WB) M (ELISA, IHC, WB) M CNTF H R H (B/N, ELISA, WB) R (B/N, ELISA, IHC, WB) H R -NGF H M R H (B/N, ELISA, IHC, WB) R (B/N, ELISA, IHC, WB) H R

A. B. Human Kidney kDa Neuro-2A kDa 200

SorLA

116 SorCS2

199

SorLA in Human Cortex. Sorting Protein-related Receptor with A-type Repeats Detection of Mouse SorLA and Human SorCS2 by Western Blot. Western blots SorCS2 in Mouse Piriform Cortex. Sortilin-related Receptor CNS Expressed 2 (SorLA) was detected in immersion-fi xed paraffi n-embedded sections of human show lysates of the Neuro-2A mouse neuroblastoma cell line and human kidney (SorCS2) was detected in immersion-fi xed frozen sections of mouse brain using a brain using a Mouse Anti-Human SorLA Monoclonal Antibody (Catalog # MAB5699). (medulla) tissue. A. The PVDF Membrane was probed with a Sheep Anti-Human/ Sheep Anti-Mouse SorCS2 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # Before incubation with the primary antibody, the tissue was subjected to heat- Mouse SorLA Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5699) AF4237). The tissue was stained using the NorthernLights™ 557-conjugated Donkey induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). followed by a HRP-conjugated Donkey Anti-Sheep IgG Secondary Antibody (Catalog Anti-Sheep IgG Secondary Antibody (Catalog # NL010; red) and counterstained with The tissue was stained using the Anti-Mouse HRP-DAB Cell & Tissue Staining Kit # HAF016). B. The PVDF Membrane was probed with a Sheep Anti-Human SorCS2 DAPI (blue). Specifi c labeling was localized to neuronal cell bodies and processes. (Catalog # CTS002; brown) and counterstained with hematoxylin (blue). Specifi c Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF4238) followed by a HRP- labeling was localized to the cytoplasmic granules. conjugated Donkey Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). SorLA and SorCS2 were detected at approximately 250 kDa and 120 kDa, respectively (as indicated).

TrkA Antibody (Pg/mL) 10-2 10-1 100 101 102

2500 2500 Cell Proliferation (Mean RFU)

2000 2000

1500 1500

1000 1000 Cell Proliferation (Mean RFU) Proliferation Cell

500 500 10-2 10-1 100 101 102 E-NGF (ng/mL)

Sortilin in Human Cingulate Cortex. Sortilin was detected in immersion-fi xed Cell Proliferation Induced by β-NGF and Neutralization by a TrkA Antibody. Phospho-APP (T668) in Alzheimer’s Disease Cortex. Phosphorylated Amyloid paraffi n-embedded sections of human brain using a Goat Anti-Human Sortilin Antigen Recombinant Human -NGF (Catalog # 256-GF) stimulates proliferation of the TF-1 Precursor Protein (APP) was detected in immersion-fi xed paraffi n-embedded sections Affi nity-purifi ed Polyclonal Antibody (Catalog # AF3154). The tissue was stained using human erythroleukemic cell line in a dose-dependent manner (gray line). Proliferation of human Alzheimer’s disease brain using a Rabbit Anti-Human/Mouse Phospho-APP the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS008; brown) and elicited by 5 ng/mL Recombinant Human -NGF is neutralized (blue line) by increasing (T668) Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF2508). The tissue counterstained with hematoxylin (blue). Specifi c labeling was localized to neuronal concentrations of a Goat Anti-Human TrkA Antigen Affi nity-purifi ed Polyclonal was stained with the Anti-Rabbit HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS005; cell bodies and processes. Antibody (Catalog # AF175). brown) and counterstained with hematoxylin (blue). Specifi c staining was localized to the cytoplasm, plasma membrane, and processes of cortical neurons.

www.RnDSystems.com/go/VPS10P 7 NEUROTROPHIC FACTORS & RECEPTORS

Additional Neurotrophic Factors & Receptors The table below provides a brief description of the neurotrophic factors that are not included within the GDNF, Neurotrophin, or VPS10P-domain families.

Table 1. Additional Neurotrophic Factors & Receptors FACTOR FULL NAME FAMILY BACKGROUND HOMOLOGY CDNF Conserved dopamine CDNF/MANF A recently discovered neurotrophic factor, CDNF promotes the survival of dopaminergic neurons in vitro and in vivo, and is a candidate 62% H M neurotrophic factor for Parkinson’s disease therapy.1 CLC/NNT/ Cardiotrophin-like cytokine/ gp130/Four helix A paralog of CNTF, CLC is secreted as a heteromeric composite cytokine with CLF-1 (CLC/CLF-1) or CNTF R (CLC/ CNTF R). Like CNTF 96% H M BSF-3 novel neurotrophin-1/ bundle cytokine and Neuropoietin, CLC signals through a tripartite complex composed of CNTF R, LIF R, and gp130 (Figure 7).2 B cell stimulating factor-3 CLF-1 Cytokine-like Factor-1 Cytokine type-I CLF-1 is secreted as a heteromeric composite cytokine with CLC (CLC/CLF-1) and signals through the tripartite complex composed of 96% H M receptor gp130, LIF R, and CNTF R (Figure 7). Recent studies suggest that Sortilin interacts with LIF R to promote CLC/CLF-1 signal transduction.3 CNTF Ciliary neurotrophic factor gp130/Four helix Originally discovered as a survival factor for chick ciliary parasympathetic neurons, recent studies showed that CNTF increased 83% H R bundle cytokine neurogenesis in mouse dentate gyrus and induced neuronal regeneration in a rat model of retinal degeneration.4,5 CT-1 Cardiotrophin-1 gp130/Four helix Highly expressed in heart, skeletal muscle, liver and kidney, with lower expression levels in brain, CT-1 has been shown to exert 79% H M bundle cytokine neuroprotective eff ects against ischemic insult by inhibiting Caspase-8 and Caspase-3.6 IL-6 Interleukin-6 IL-6 family/Four helix IL-6 aff ects a range of important actions in the CNS, including eff ects on neuron homeostasis, astrogliogenesis, and neuronal 41% H M bundle cytokine diff erentiation.7 LIF Leukemia inhibitory factor gp130/Four helix LIF is a variably glycosylated polypeptide originally identifi ed as a proliferation inhibitor and diff erentiation inducer of eth mouse M1 78% H M bundle cytokine myeloid leukemia cell line.8 MANF Mesencephalic astrocyte- CDNF/MANF Recent studies of this neuroprotective factor suggest the 3D structure of the C-terminal domain is homologous to the SAP domain of 99% H R derived neurotrophic factor Ku70, an established inhibitor of pro-apoptotic Bcl-2-associated X protein (Bax).9 98% H M Meteorin – Orphan ligand A developmentally important secreted protein that is expressed in undiff erentiated neural precursor cells and astrocyte lineages. 82% H M GFAP-positive glial diff erentiation induced by Meteorin is thought to require activation of the Jak-STAT3 pathway.10 MK Midkine Heparin-binding Midkine promotes the survival and migration of many cell types and has been pathologically linked to a variety of cancers. Recent 87% H M growth factor studies showed that increased Midkine expression in rat brain was neuroprotective against excitotoxic and ischemic injury.11,12 Neuritin Also known as Candidate – Neuritin is a GPI-linked molecule that promotes neurite extension and arborization.13 The expression of Neuritin is upregulated 99% H M R plasticity gene 15 (CPG15) in response to BDNF, NT-3, androgens, and hypoxia.14 NP Neuropoietin, Cardiotrophin-2 gp130/Four helix Highly expressed in neuroepithelia, Neuropoietin is an important factor for CNS development. Recent studies suggest Neuropoietin 95% M R (CT-2) bundle cytokine can signal independently of LIF R in non-neuronal cells.15 PTN Pleiotrophin/heparin-binding Heparin-binding A developmentally regulated neurotrophic factor, Pleiotrophin can be used as an attachment substrate to promote neurite outgrowth. 98% H M R neurotrophic factor (HBNF) growth factor Studies showed that Pleiotrophin knockout increased the vulnerability of mouse striatal dopaminergic neurons to amphetamine neurotoxicity.16 Teneurin-1 – Tenascin A single transmembrane protein, Teneurin-1 promotes neurite outgrowth and cell-cell adhesion via homophilic interactions. 96% H M C-terminal cleavage generates Teneurin C-terminal associated peptide (TCAP-1), which has been shown to increase neurite outgrowth (a.a. 1-317) in immortalized mouse hypothalamic neurons.17

KEY: H: Human M: Mouse R: Rat

References

1. Lindholm, P. et al. (2007) Nature 448:73.

CNTF 2. Zou, X. et al. (2009) Vet. Pathol. 46:514. 3. Larsen, J.V. et al. (2010) Mol. Cell. Biol. 30:4175.

4. Blanchard, J. et al. (2010) J. Alzheimer’s Dis. 21:1185.

5. Li, Y. et al. (2010) PLoS One 5: e9495. Extracellular 6. Peng, H. et al. (2010) J. Neurosci. Res. 88:1041.

Plasma Membrane 7. Spooren, A. et al. (2011) Brain Res. Rev. 81:1004.

CNTF RD 8. Gearing, D.P. et al. (1987) EMBO J. 6:3995. gp130 Intracellular LIF R Sortilin PI 3-K/Akt Pathway 9. Hellman, M. et al. (2010) J. Biol. Chem. 4:215. 10. Lee, H.S. et al. (2010) J. Cell. Sci. 123:1959. CNTF Internalization JAK/STAT Pathway Ras/MAPK/ERK 11. Kim, Y.B. et al. (2010) BMC Neurosci. 11:42.

12. Ishikawa, E. et al. (2009) J. Neurol. Sci. 285:78.

13. Javaherian, A. & H.T. Cline (2005) Neuron 45:505.

Domain Key: VPS10P Fibronectin type III Cytokine receptor homology Ig-like GPI anchor 14. Fargo, K.N. et al. (2008) J. Neurotrauma 25:561.

Figure 7. CNTF Signaling. CNTF homodimers bind to GPI-linked CNTF R, which recruits the signaling subunits gp130 and 15. White, U.A. & J.N. Stephens (2010) Biochem. Biophys. Res. Commun. 395:48. LIF R. Recent studies discovered that Sortilin binds to CNTF and interacts with LIF R to facilitate CNTF internalization and signal 16. Gramage, E. et al. (2010) Neuroscience 170:308. transduction, respectively.3 A similar tripartite complex governs signaling of CLC/CLF-1 and CLC/CNTF R heteromeric composite cytokines. The cytokine receptor homology domains contain characteristic cysteine residues and a WSXWS pentapeptide motif. 17. Al Chawat, A. et al. (2007) Neuroscience 140:1241.

8 For research use only. Not for use in diagnostic procedures. NEUROTROPHIC FACTORS & RECEPTORS

R&D Systems Products for Additional Neurotrophic Family Signaling Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Cardiotrophin-1/CT-1 H M H (B/N, WB) M (B/N, ELISA, WB) M LIF R H M H (B/N, FC, WB)

CDNF H M H (IHC, WB) M (IHC, WB) MANF HH (WB)

CLC HH (WB) Meteorin MM (ELISA, IHC, WB) M CLC/CNTF R Chimera H Meteorin-like/METRNL M

CLF-1 H Midkine HH (IHC, WB)

CNTF H R H (B/N, ELISA, WB) R (B/N, ELISA, IHC, WB) H R Neudesin H (IHC)

gp130 H M R H (B/N, ELISA, FC, WB) M (B/N, ELISA, FC, IHC, WB) H M Neuropoietin/NP MM (WB)

IL-6 H M R Ca H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M R Ca Pleiotrophin/PTN H M H (WB) CR E F P R (B/N, ELISA, IHC, WB) Ca (B/N, ELISA, FC, IHC, WB) CR (B/N, WB) CR F P E (B/N, IHC, WB) F (B/N, IHC, WB) P (B/N, ELISA, FC, IHC, WB) Teneurin-1 H (WB)

LIF H (B/N, ELISA, FC, IHC, WB) M (B/N, IHC, WB) H M

Mouse gp130 Antibody (Pg/mL) 10-2 10-1 100 101 20000 20000 Cell Proliferation (Mean CPM)

15000 15000

10000 10000

5000 5000 Cell Proliferation (Mean CPM) Proliferation Cell

0 0 10-1 100 10-1 10-2 10-3 Mouse gp130 (ng/mL)

CDNF in Human Cortex. Conserved Dopamine Neurotrophic Factor (CDNF) was IL-6 Inhibition of Cell Proliferation and Neutralization by a gp130 Antibody. Meteorin in Diﬀ erentiated Rat Cortical Stem Cells. Meteorin was detected in detected in immersion-fi xed paraffi n-embedded sections of human cortex using Recombinant Human IL-6 (Catalog # 206-IL) inhibits proliferation in the M1 mouse immersion-fi xed 7 day diff erentiated rat cortical stem cells using a Rat Anti-Mouse a Goat Anti-Human CDNF Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # myeloid leukemia cell line in a dose-dependent manner (green line). Activity Meteorin Monoclonal Antibody (Catalog # MAB3475). The cells were stained with AF5097). Before incubation with the primary antibody, the tissue was subjected to elicited by Recombinant Human IL-6 (20 ng/mL) is neutralized (gray line) by the NorthernLights™ 557-conjugated Goat Anti-Rat IgG Secondary Antibody heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # increasing concentrations of a Goat Anti-Mouse gp130 Antigen Affi nity-purifi ed (Catalog # NL013; red) and counterstained with DAPI (blue). CTS013). The tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Polyclonal Antibody (Catalog # AF468). Kit (Catalog # CTS008; brown) and counterstained with hematoxylin (blue).

Phospho-gp130 1000 500 250 125 62.5 IMR-32 HEK293 kDa kDa Mouse Brain 150 Lysate (Pg) 1.4 Teneurin-2 120 1.2 98 1.0 90 0.8

Teneurin-1 60 0.6

55 Number Cell Relative 0.4

30 Phospho-gp130 (O.D.) 199 0.2

37 0 0 100 101 102 103 104 100 50 25 12.5 6.25 3.13 1.56 IL-6 RD Lysate (Pg)

Detection of Human Teneurin-1 and Mouse Teneurin-2 by Western Blot. Western Detection of IL-6 R by Flow Cytometry. The U937 human histiocytic lymphoma cell Measurement of Phospho-gp130 Levels using the DuoSet® IC ELISA. Lysates were blots show lysates of the IMR-32 human neuroblastoma cell line, the HEK293 line was stained with a Mouse Anti-Human IL-6 RMonoclonal Antibody (Catalog # prepared from the HepG2 human hepatocellular carcinoma cell line treated with

human embryonic kidney cell line, and mouse brain. A. The PVDF membrane was MAB227, fi lled histogram) or a Mouse IgG1 Isotype Control (Catalog # MAB002, open 100 ng/mL Recombinant Human IL-6 (Catalog # 206-IL) and 200 ng/mL probed with a Sheep Anti-Human Teneurin-1 Antigen Affi nity-purifi ed Polyclonal histogram), followed by a PE-conjugated Goat Anti-Mouse IgG Secondary Antibody Recombinant Human IL-6 Receptor  (Catalog # 227-SR). Lysates were serially Antibody (Catalog # AF6324) followed by a HRP-conjugated Donkey Anti-Sheep IgG (Catalog # F0102B). diluted and Phospho-gp130 levels were quantifi ed using the Human Phospho- Secondary Antibody (Catalog # HAF016). B. The PVDF membrane was probed with gp130 DuoSet IC ELISA (Catalog # DYC3407). gp130 was immunoprecipitated from a Sheep Anti-Human/Mouse/Rat Teneurin-2 Antigen Affi nity-purifi ed Polyclonal the same lysates using an anti-gp130 monoclonal antibody, and immunoblotted Antibody (Catalog # AF4578) followed by a HRP-conjugated Donkey Anti-Sheep IgG (inset). Western blots were incubated with a Mouse Biotinylated Anti-Phospho- Secondary Antibody (Catalog # HAF016). Teneurin-1 and Teneurin-2 were detected Tyrosine Monoclonal Antibody (Catalog # BAM1676) to detect phospho-gp130. at approximately 65 kDa and 300 kDa, respectively (as indicated).

www.RnDSystems.com/go/NeurotrophicFactors 9 NEURAL DEVELOPMENT & AXON GUIDANCE

Ephrin Ligands & Eph Receptors Highly expressed in the developing nervous system, Ephrins are ligands signaling.9 Ephrin-B ligands have also been shown to promote synapse for transmembrane Eph receptor tyrosine kinases. They are involved in a formation and maturation by reverse signaling following binding by wide range of critical developmental processes, including cell adhesion, EphB2.10 cell migration, angiogenesis, axon guidance, and synaptogenesis.1 In Many studies have investigated the modulation of NMDA receptor addition, Ephrin/Eph signaling is also important for neuronal regeneration expression by EphB receptors, an action that is thought to aff ect learning and neural progenitor cell diff erentiation in the adult.2 There are nine and memory. Recent studies by Nolt et al. showed that EphB2 phos- Ephrin ligands, which are divided into two subfamilies, Ephrin-A1 to -A6 phorylates NMDA receptor subunit NR2B to control its localization at the and Ephrin-B1 to -B3. Ephrin-A ligands are glycosylphosphatidylinositol synapse.11 Further studies in a transgenic mouse model of Alzheimer’s (GPI)-linked proteins that generally bind to EphA receptors. Ephrin-B disease suggest that amyloid- induced impairments in long-term ligands are transmembrane proteins that contain a cytoplasmic region potentiation and memory require EphB2-dependent downregulation of and bind to EphB receptors. Exceptions to this general rule are Ephrin-A5, NMDA receptors.12 Recent research also suggests that Ephrin B ligands are which at high concentrations will bind to EphB2, and Ephrin-B ligands critical for Reelin-induced neuronal migration during the development of which bind to EphA4 (Table 2). laminated structures in the brain.13 Binding of Ephrin ligands triggers Eph receptor clustering, autophos- References phorylation, and induction of signaling pathways that induce cytoskeletal 1. Pasquale, E.B. et al. (2008) Cell 133:38. restructuring and modifi cation of cell adhesion. A unique feature of Ephrin/Eph signaling is that Ephrin ligands can also act as receptors and 2. Parrinello, S. et al. (2010) Cell 143:145. Eph receptors can function as ligands (Figure 8). To clarify this bidirectional 3. Xu, N.J. et al. (2009) Nat. Neurosci. 12:268. relationship, an Ephrin ligand binding to an Eph receptor induces forward 4. Naska, S. et al. (2010) Mol. Cell. Neurosci. 45:108. signaling that is dependent on a catalytically active intracellular Eph 5. Lim, Y.S. et al. (2008) Neuron 59:746. kinase domain. In parallel, Ephrin ligands can also reverse signal into their 6. Klein, R. (2009) Nat. Neurosci. 12:15. host cell.3 This action requires the association of GPI-linked Ephrin-A with a transmembrane receptor, or modifi cation of the Ephrin-B cytoplasmic 7. Penzes, P. et al. (2003) Neuron 37:263. region. For example, Ephrin-A-induced axon repulsion and Ephrin-B3 8. Tolias, K.F. et al. (2007) Proc. Natl. Acad. Sci. USA 104:7265. -dependent growth cone collapse have both been shown to require 9. Bourgin, C. et al. (2007) J. Cell. Biol. 178:1295. reverse signaling pathways.4,5 10. Kayser, M.S. et al. (2006) J. Neurosci. 26:12152.

Recent research has focused on the ability of Ephrin/Eph signaling to 11. Nolt, M.J. et al. (2011) J. Neurosci. 31:5353. regulate synapse formation and synaptic plasticity.6 Through forward 12. Cissé, M. et al. (2011) Nature 469:47. signaling pathways, EphB2 has been shown to control postsynaptic dendritic spine formation via Rho family GTPases and the guanine- 13. Sentürk, M. et al. (2011) Nature 472:356. nucleotide exchange factor TIAM1.7,8 In contrast, Ephrin-A3 stimulation 14. Surawska, H. et al. (2004) Cytokine Growth Factor Rev. 15:419. of EphA4 regulates dendritic spine morphology by inhibiting Integrin 1

Table 2. LIGAND BINDING SPECIFICITIES Axon Repulsion Axon Terminal REVERSE Eph Receptors Ephrin Ligands Synapse Formation & Maturation SIGNALING EphA1 Ephrin-A1 (low); Ephrin-B1 EphA2 Ephrin-A1, -A3, -A4, -A5 EphA3 Ephrin-A2, -A3, -A4, -A5 Ephrin-A3 Ephrin-B2 EphA4 Ephrin-A1, -A4, -A5; -B2; Ephrin-A2, -A3 (low) Association with a BIDIRECTIONAL transmembrane receptor SIGNALING EphA5 Ephrin-A1, -A2, -A3, -A4, -A5 EphA6 Ephrin-A1 EphA7 Ephrin-A1, -A2, -A3, -A4, -A5 EphA8 Ephrin-A1, -A2, -A3, -A4, -A5 FORWARD EphA10 Unknown EphA4 EphB2 SIGNALING Dendrite EphB1 Ephrin-B1, -B2, -B3 Reduced Spine Length Increased Spine EphB2 Ephrin-A5 & Density Formation EphB3 Ephrin-B1, -B2 EphB4 Ephrin-B2 Domain Key: GPI-linked Globular Cysteine-rich Fibronectin type III EphB5 Unknown Tyrosine kinase Sterile D motif PDZ-binding Death domain EphB6 Unknown

Figure 8. Bidirectional Signaling by Ephrin Ligands and Eph Receptors. Illustration depicts examples of forward and reverse Table 2. Ligand Binding Speciﬁ cities for Eph Family Receptors. The ligand binding specifi cities for diff erent Eph receptors signaling in neurons. are shown.14

10 For research use only. Not for use in diagnostic procedures. NEURAL DEVELOPMENT & AXON GUIDANCE

R&D Systems Products for Ephrin Ligands & Eph Receptors RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Ephrin-A EphA4 MM (IHC, WB)

Ephrin-A1 H M M (WB) EphA5 H M R H (WB) M (FC, IHC, WB) R (FC, IHC, WB) H M

Ephrin-A2 MM (B/N, IHC, WB) EphA6 H M M (B/N, WB)

Ephrin-A3 HH (WB) EphA7 H M M (WB)

Ephrin-A4 H M H (WB) M (WB) EphA8 MM (WB)

Ephrin-A5 HH (IHC, WB) EphA10 HH (IHC) Ephrin-B EphB

Ephrin-B H (WB) M (WB) R (WB) Ch (WB) X (WB) EphB1 RR (FC, IHC, WB)

Ephrin-B1 MM (IHC, WB) EphB2 H M H (FC, IHC, WB) M (FC, IHC, WB)

Ephrin-B2 M Z M (FC, IHC, WB) Z (WB) EphB3 H M H (WB) M (FC, IHC, WB)

Ephrin-B3 HH (IHC, WB) EphB4 H M H (FC, IHC, WB) M (FC, IHC, WB) H

EphA EphB6 H M H (FC, WB) M (WB)

EphA1 H M H (FC, IHC, WB) M (FC, IHC, WB) H Eph/Ephrin Sampler Packs

EphA2 H M H (FC, IHC, WB) M (FC, IHC, WB) H Eph H M R

EphA3 H M M (IHC, WB) Ephrin H M

Species Key: H Human M Mouse R Rat Ch Chicken X Xenopus Z Zebrafi sh Application Key: B/N Blocking/Neutralization FC Flow Cytometry IHC Immunohistochemistry WB Western blot

Phospho-EphA1 50 25 12.5 6.25 3.13 2.5 Lysate (Pg) 1.2 Untreated Ephrin A1 + Fc 2.0 1.0 Phospho-EphA5

1.5 0.8 EphA5 0.6 1.0 0.4

Phospho-EphA1 (O.D.) 0.5 Phospho-EphA5 (O.D.) 0.2 0 12.5 6.25 3.13 1.56 0.78 0.39 0 Lysate (Pg) Untreated Ephrin A1 + Fc

Measurement of Phospho-EphA1 Levels using the DuoSet IC ELISA. Lysates Ephrin-A2 in Rat Hippocampal Neurons. Ephrin-A2 was detected in immersion- Measurement of Phospho-EphA5 Levels using the DuoSet® IC ELISA. Lysates prepared from HEK293 human embryonic kidney cells transfected with human fi xed rat hippocampal neurons using a Goat Anti-Mouse Ephrin-A2 Antigen Affi nity- prepared from HEK293 human embryonic kidney cells transfected with human EphA1, were untreated or treated with 3.0 g/mL Recombinant Mouse Ephrin-A2 purifi ed Polyclonal Antibody (Catalog # AF603). Neurons were stained (green) and EphA5, were untreated or treated with 3.0 g/mL Recombinant Mouse Ephrin-A1

Fc Chimera (Catalog # 603-A2) and 0.3 g/mL Recombinant Human IgG1 (Catalog glial cells were labeled by using an anti-GFAP antibody (red). Fc Chimera (Catalog # 602-A1) and 0.3 g/mL Goat Anti-Human IgG Fc Affi nity- # 110-HG) for 20 minutes. Lysates were serially diluted and Phospho-EphA1 levels purifi ed Polyclonal Antibody (Catalog # G-102-C) for 20 minutes. Phospho-EphA5 were quantifi ed using the Human Phospho-EphA1 DuoSet IC ELISA (Catalog # levels were quantifi ed using the Human/Mouse Phospho-EphA5 DuoSet IC ELISA DYC4835). EphA1 was immunoprecipitated from the same lysates using an anti- (Catalog # DYC5037). EphA5 was immunoprecipitated from the same lysates using EphA1 monoclonal antibody, and immunoblotted (inset). Western blots were an anti-EphA5 polyclonal antibody, and immunoblotted (inset). Western blots incubated with a Mouse HRP-conjugated Anti-Phospho-Tyrosine Monoclonal were incubated with a Mouse HRP-conjugated Anti-Phospho-Tyrosine Monoclonal Antibody (Catalog # HAM1676) to detect phospho-EphA1. Antibody (Catalog # HAM1676) to detect phospho-EphA5. Blots were stripped and total EphA5 was detected using a Goat Anti-Rat EphA5 Biotinylated Affi nity-purifi ed Polyclonal Antibody (Catalog # BAF541). kDa 198 Mouse Brain 116 EphB2 84

54 37 29

EphB2 in Embryonic Mouse Brain. EphB2 was detected in immersion-fi xed frozen Detection of Mouse EphB2 by Western Blot. Western blot shows lysates of EphA2 in Mouse Neural Tube. EphA2 was detected in perfusion-fi xed frozen sections of embryonic mouse brain (15 d.p.c.) using a Goat Anti-Mouse EphB2 mouse brain tissue. The PVDF membrane was probed with a Rat Anti-Mouse sections of mouse embryo (11 d.p.c.) using a Goat Anti-Mouse EphA2 Antigen Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF467). The tissue was EphB2 Monoclonal Antibody (Catalog # MAB4672) followed by a HRP-conjugated Affi nity-purifi ed Polyclonal Antibody (Catalog # AF639). The tissue was stained stained with the NorthernLights™ 557-conjugated Donkey Anti-Goat IgG Secondary Goat Anti-Rat IgG Secondary Antibody (Catalog # HAF005). EphB2 was detected at using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS008; brown) Antibody (Catalog # NL001; red) and counterstained (green). approximately 106 kDa (as indicated). and counterstained with hematoxylin (blue). www.RnDSystems.com/go/Ephrins 11 NEURAL DEVELOPMENT & AXON GUIDANCE

Hedgehog Family In vertebrates, the hedgehog family consists of three secreted transcriptional activator and GLI-3, a transcriptional repressor. In the morphogens, Desert Hedgehog (Dhh), Indian Hedgehog (Ihh), and Sonic absence of Hedgehog, Suppressor of Fused (SuFu) retains Gli transcription Hedgehog (Shh), which are critical for a range of biological processes, factors in the cyto plasm and promotes processing of Gli-A into the including neurogenesis, neural diff erentiation, tissue patterning, axon truncated repressor form.7 guidance, and synaptogenesis.1,2 Produced as large precursors, Hedgehog Transmission of Shh signaling is governed by a series of additional proteins undergo autoproteolysis to yield a biologically active N-terminal proteins. Ptc negatively regulates Shh signal transduction by inducing domain, which is further modifi ed by the addition of palmitate and rapid internalization and degradation of Shh.8 In addition, Hedgehog- cholesterol.3 Subsequent secretion of mature Hedgehog is dependent on interacting protein (Hip), a glycoprotein, blocks Shh signaling by binding Dispatched, a twelve-pass transmembrane protein.4 to and sequestering Shh at the cell membrane.9 In contrast, the cellular The major receptor for Hedgehog proteins is a complex of Patched (Ptc) eff ects of Shh are augmented following binding of Shh to the membrane and Smoothened (Smo). Ptc is a twelve-pass transmembrane protein that proteins CAM-related/downregulated by oncogenes (CDO) and Brother displays a transporter-like structure and binds Hedgehog ligands. Smo is of CDO (BOC).10 Recent studies suggest that the positive eff ect of a seven-pass transmembrane putative G protein-coupled receptor that CDO is further enhanced through cooperation with Gas1, a glycosyl- transmits a downstream signal. In the absence of ligand, Ptc represses the phosphatidylinositol (GPI)-linked plasma membrane protein.11 activity of Smo. Following Hedgehog binding, Ptc repression of Smo is References relieved and signaling cascades promote the transcription of target genes 1. Traiff ort, E. et al. (2009) J. Neurochem. 113:576. (Figure 9). 2. Sasaki, N. et al. (2010) Mol. Cell. Neurosci. 45:335. Hedgehog signaling occurs at the primary cilium, an organelle that 3. Farzan, S.F. et al. (2008) AJP Gastrointest. Liver Physiol. 294:9844. emanates from the cell body into the extracellular environment.5 Activation of target genes by Hedgehog is induced via the glioma- 4. Etheridge, L.A. et al. (2010) Development 137:133. associated oncogene homolog (Gli) family of transcription factors. The 5. Tukachinsky, H. et al. (2010) J. Cell. Biol. 191:415. three Gli proteins each contain fi ve zinc-fi nger DNA-binding domains 6. Machold, R. et al. (2003) Neuron 39:937. and have variable N-terminal domains. Hedgehog proteins inhibit the 7. Humke, E.W. et al. (2010) Genes Dev. 24:670. cleavage of Gli into their transcriptional repressor forms (Gli-R) and promote the nuclear translocation of Gli transcriptional activators (Gli-A). 8. Incardona, J.P. et al. (2000) Proc. Natl. Acad. Sci. USA 97:12044. GLI-1 is classically used as a marker for Hedgehog pathway activation, 9. Bishop, B. et al. (2009) Nat. Struct. Mol. Biol. 16:698. but is considered more of an amplifi cation factor than a transduction 10. Kavran, J.M. et al. (2010) J. Biol. Chem. 285:24584. 6 element. The majority of Hedgehog signaling is mediated by GLI-2, a 11. Martinelli, D.C. & C.M. Fan (2009) J. Biol. Chem. 284:19169.

Signal NC Hedgehog Preprotein Palmitate Autoproteolysis Cholesterol

TARGET CELL Dispatched (Absence of Ligand) TARGET CELL HIP (Presence of Ligand)

Hedgehog Multimer Patched Patched CDO Smoothened BOC Gas1 Lysosome Smoothened Gli-R SuFu GPI Anchor Gli-A Gli-R PKA Gli-A

SuFu Transcriptional Activation of Gli-R Repression Gli-A Target Genes

Nucleus Nucleus

Figure 9. The Hedgehog Signaling Pathway. In a signaling cell, Hedgehog preprotein undergoes autoproteolysis, followed by the addition of cholesterol and palmitate before Dispatched-dependent secretion and extracellular multimerization. In the absence of Hedgehog ligand (left), Patched suppresses Smoothened and Suppressor of Fused (SuFu) retains full length Gli (Gli-A) in the cytoplasm, where it is cleaved to form a transcriptional repressor (Gli-R). In the presence of Hedgehog (right), Patched suppression of Smoothened is relieved, the actions of SuFu are attenuated, and Gli-A translocates to the nucleus to activate target genes.

12 For research use only. Not for use in diagnostic procedures. NEURAL DEVELOPMENT & AXON GUIDANCE

R&D Systems Products for Hedgehog Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Hedgehogs GLI-2 H (ChIP, IHC, WB) M (IHC, WB) H

Desert Hedgehog/Dhh H M M (IHC, WB) GLI-3 H (ChIP, IHC, WB) M (ChIP, IHC, WB) H

Indian Hedgehog/Ihh H M H (WB) M (IHC, WB) Glypican 3 H M H (FC, IHC, WB)

Sonic Hedgehog/Shh H M H (FC, WB) M (B/N, ELISA, FC, IHC, WB) M GSK-3/ H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R

Hedgehog Related Molecules & Regulators GSK-3 H (IHC, WB) M (IHC, WB) R (IHC, WB) H

BOC HH (WB) M (IHC, WB) GSK-3 HH (FC, IHC, WB) M (FC, WB) R (FC, WB) H M R

CDO HH (FC, IHC, WB) M (IHC, WB) Hip MM (WB) M

DISP1 H (WB) LIN-41 H (IHC, WB)

Gas1 H M H (ELISA, WB) M (ELISA, IHC, WB) H M Patched 1/PTCH M (FC, IHC, WB)

GLI-1 H (ChIP, IHC, WB) M (IHC, WB) H Patched 2/PTCH2 H (FC, IHC, WB)

Sonic Hedgehog Antibody (Pg/mL) 10-1 100 101 102 Alkaline PhosphataseAlkaline (Mean O.D.)

1.6 1.6

1.2 1.2 Anti-GLI-3 0.8 0.8 No Antibody IgG bcl-2 gli-1

0.4 0.4 500 bp Alkaline Phosphatase (Mean O.D.) Alkaline Phosphatase 0.0 0.0 10-2 10-1 100 101 Sonic Hedgehog (Pg/mL)

Alkaline Phosphatase Production Induced by Sonic Hedgehog and Patched 1 in Mouse Spinal Cord. Patched 1 was detected in immersion-fi xed Detection of GLI-3 Binding to the gli-1 Promoter by Chromatin Immuno- Neutralization by a Sonic Hedgehog Antibody. Recombinant Mouse Sonic frozen sections of mouse spinal cord using a Rat Anti-Mouse Patched 1 Monoclonal precipitation. Jurkat human leukemic T cells were stimulated for 30 minutes with Hedgehog N-Terminus (Catalog # 461-SH) induces alkaline phosphatase production Antibody (Catalog # MAB41051). The tissue was stained (red) and counterstained 50 ng/mL PMA and 200 ng/mL ionomycin, fi xed, and lysed. Binding of GLI-3 to in the C3H10T1/2 mouse embryonic fi broblast cell line in a dose-dependent manner with DAPI (blue). Specifi c staining has localized to the fl oor plate. the gli-1 promoter was assessed using the ExactaChIP™ Human GLI-3 Chromatin (orange line). Alkaline phosphatase production elicited by Recombinant Mouse Immunoprecipitation Kit (Catalog # ECP3690). Briefl y, cell lysates were incubated Sonic Hedgehog N-Terminus (5 g/mL) is neutralized (gray line) by increasing with a biotinylated goat anti-human GLI-3 affi nity-purifi ed polyclonal antibody or concentrations of a Rat Anti-Mouse Sonic Hedgehog N-Terminus Monoclonal a biotinylated goat IgG affi nity-purifi ed polyclonal antibody (both provided in the Antibody (Catalog # MAB4641). kit), followed by MagCellect™ Streptavidin Ferrofl uid (Catalog # MAG999). DNA was purifi ed from the immunoprecipitates, and thegli-1 promoter, which is regulated by GLI-3, was detected by standard PCR using primers provided in the kit. The bcl-2 promoter was used as a negative control.

kDa Mouse Embryonic Fibroblasts

203 Patched 1 117

Sonic Hedgehog in Mouse Spinal Cord. Sonic Hedgehog was detected in Detection of Mouse Patched 1 by Western Blot. Western blot shows lysates of Patched 2 in Human Basal Cell Carcinoma. Patched 2 was detected in immersion- immersion-fi xed frozen sections of mouse spinal cord using a Goat Anti-Mouse mouse embryonic fi broblasts. The PVDF membrane was probed with a Rat Anti- fi xed paraffi n-embedded sections of human basal cell carcinoma using a Goat Sonic Hedgehog C-Terminus Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog Mouse Patched 1 Monoclonal Antibody (Catalog # MAB41051) followed by a HRP- Anti-Human Patched 2 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # # AF445). The tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining conjugated Goat Anti-Rat IgG Secondary Antibody (Catalog # HAF005). Patched 1 AF4078). Before incubation with the primary antibody, the tissue was subjected Kit (Catalog # CTS008; brown) and counterstained with hematoxylin (blue). was detected at approximately 160 kDa (as indicated). to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). The tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS008; brown) and counterstained with hematoxylin (blue). Specifi c labeling was localized to the cytoplasm of keratinocytes.

www.RnDSystems.com/go/Hedgehog 13 NEURAL DEVELOPMENT & AXON GUIDANCE

Netrin Family Netrins are a highly conserved family of chemotropic guidance cues via their cytoplasmic DCC-binding and P1 domains. The mechanism of that infl uence axon guidance, neuronal migration, and cell survival.1,2 In Netrin-1-induced UNC5-dependent repulsion may involve remodeling vertebrates, members include the secreted proteins Netrin-1, -2, and -4 of the actin cytoskeleton by RhoA and Rac1 GTPases.6 Additional studies (Netrins), and Netrin-G1 and -G2 (Netrin-Gs), which are glycosylphospha- suggest that Netrin-1 also mediates axon guidance through binding tidylinositol (GPI)-linked to the plasma membrane. During embryogenesis, to Down’s Syndrome Cell Adhesion Molecule (DSCAM), and promotes netrins direct axonal growth cones to their cellular targets, where synapses interneuron migration following interactions with integrins. 7,8 are formed. Netrins are known to be bifunctional guidance cues that can Netrin-Gs do not bind to DCC or UNC5. Instead they interact with Netrin-G steer axonal growth through both attractive and repulsive mechanisms. Ligands (NGLs) that are predominantly located at the postsynaptic side This functional dichotomy is achieved through ligand-dependent of excitatory synapses (Figure 11). Presynaptic Netrin-G1 and -G2 bind formation of distinct receptor complexes that transduce specifi c signaling to postsynaptic NGL-1 and NGL-2, respectively. In addition, recently cascades to promote attraction or repulsion. discovered NGL-3 binds to presynaptic Leukocyte Antigen-related (LAR).9 Netrins bind to two major receptor families, the Deleted in Colorectal All three interactions are thought to couple synaptic adhesion with the Cancer (DCC) subfamily and the UNC5 homolog family (UNC5H1-4). DCC bidirectional assembly of synaptic proteins, including PSD-95 and NMDA receptors are type 1 transmembrane proteins with four immunoglobulin receptors.10,11 (Ig)-like and six fi bronectin type III repeats in the extracellular domain and References three conserved intracellular domains (P1-3). Netrin binding is believed to 1. Cirulli, V. & M. Yebra (2007) Nat. Rev. Mol. Cell. Biol. 8:296. require the fourth and fi fth fi bronectin type-III repeats.3 UNC5 receptors 2. Rajasekharan, S. & T.E. Kennedy (2009) Genome Biol. 10:239. are type I transmembrane proteins that contain two extracellular Ig-like domains, which are required for Netrin binding, and two thrombospondin- 3. Geisbrecht, B.V. et al. (2003) J. Biol. Chem. 278:32561. like domains. The intracellular region includes a zonula-occludens 5 (ZU5) 4. Ma, W. et al. (2010) Structure 18:1502. domain, a DCC binding domain, and a death domain. 5. Tcherkezian, J. et al. (2010) Cell 141:632. Netrin-1 functions as an attractant by binding to DCC and promoting 6. Picard, M. et al. (2009) Cell Signal. 21:1961. DCC clustering through interactions between the P3 domains (Figure 10). 7. Andrews, G.L. et al. (2008) Development 135:3839. Recent studies suggest the underlying signaling pathway may involve 8. Stanco, A. et al. (2009) Proc. Natl. Acad. Sci. USA 106:7595. ERK2 and association of DCC with the translational machinery.4,5 Netrin-1 exerts short-range repulsion, in close proximity to the secreting cell, by 9. Woo, J. et al. (2009) Nat. Neurosci. 12:428. binding to homodimers of UNC5. In addition, Netrin-1 acts as a long- 10. Woo, J. et al. (2009) Mol. Cell. Neurosci. 42:1. range repellent through heterodimers of UNC5 and DCC, which associate 11. Kwon, S.K. et al. (2010) J. Biol. Chem. 285:13966.

DSCAM Presynaptic Postsynaptic UNC5/DCC Heterodimer DCC Homodimer

UNC5 Homodimer Netrin Netrin-G1 NGL-1

Netrin-G2 NGL-2 Extracellular

Plasma Membrane

11 Intracellular 1 LAR 22 2 NGL-3 3 3 3

ATTRACTION REPULSION

Domain Key: Ig-like Fibronectin type III Conserved IC Laminin-like EGF repeat Heparin-binding Pak-interacting Thrombospondin-like ZU5 DCC-binding Death domain GPI anchor Cysteine-rich Leucine-rich PDZ-binding Phosphatase

Figure 10. Netrins Exert Bifunctional Guidance Cues. Following binding to DCC or DSCAM, Netrins promote axon guidance Figure 11. Netrin-G Ligands Promote Synaptic Adhesion. At excitatory synapses, presynaptic Netrin-G1, Netrin-G2, and through chemoattractant signals. In the presence of UNC5, Netrins exert a repulsive cue via UNC5 homodimers (short-range) or Leukocyte Antigen-Related (LAR) interact with postsynaptic Netrin G ligands, NGL-1, NGL-2, and NGL-3, respectively. These UNC5/DCC heterodimers (long-range). molecular interactions are believed to promote the assembly of synaptic proteins following synaptic adhesion.

14 For research use only. Not for use in diagnostic procedures. NEURAL DEVELOPMENT & AXON GUIDANCE

R&D Systems Products for Netrin Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs Netrins Netrin Receptors

Netrin-1 H M Ch M (B/N, WB) Ch (WB) DCC MH (IHC, WB) M (B/N, WB)

Netrin-2 Ch Ch (B/N, WB) DSCAM HH (FC, IHC, WB)

Netrin-4 H M H (IHC, WB) M (B/N, IHC, WB) DSCAM-L1 HH (FC, WB)

Netrin-G1a MM (WB) LAR H (WB) M (WB) R (WB)

Netrin-G2a MM (IHC, WB) Neogenin MM (B/N, IHC, WB)

NGL-1/LRRC4C HH (WB) Nope MM (ELISA, WB) M

Species Key: H Human M Mouse R Rat Ch Chicken UNC5H1 H R R (B/N, IHC, WB) Application Key: B/N Blocking/Neutralization ELISA ELISA Capture and/or Detection FC Flow Cytometry IHC Immunohistochemistry WB Western blot UNC5H2 RR (IHC, WB)

UNC5H3 HH (IHC, WB)

UNC5H4 HH (WB)

kDa CHP-100 80 261 70 Full Length LAR 172 60 Cleaved LAR ECD 50

40 97 30

Relative Cell Number Cell Relative 20 42 10 0 100 101 102 103 104 DSCAM-L1 21

Netrin-4 in Mouse Cerebellum. Netrin-4 was detected in perfusion-fi xed frozen Detection of Human DSCAM-L1 by Flow Cytometry. Down Syndrome Cell Detection of Human LAR by Western Blot. Leukocyte Antigen-related (LAR) sections of mouse brain using a Goat Anti-Mouse Netrin-4 Antigen Affi nity-purifi ed Adhesion Molecule (DSCAM) long isoform was detected in the A172 human tyrosine phosphatase was detected in lysates of the CHP-100 human neuroblastoma Polyclonal Antibody (Catalog # AF1132). The tissue was stained with the Anti-Goat glioblastoma cell line using a Goat Anti-Human DSCAM-L1 Antigen Affi nity-purifi ed cell line. The PVDF membrane was probed with a Rat Anti-Human LAR Monoclonal HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS008; brown) and counterstained Polyclonal Antibody (Catalog # AF3315, fi lled histogram) or a Goat IgG Isotype Antibody (Catalog # MAB3004) followed by a HRP-conjugated Goat Anti-Rat IgG with hematoxylin (blue). Specifi c staining was localized to endothelial cells in the Control (Catalog # AB-108-C, open histogram), followed by a PE-conjugated Donkey Secondary Antibody (Catalog # HAF005). LAR was detected at approximately 207 vasculature. Anti-Goat IgG Secondary Antibody (Catalog # F0107). kDa and 139 kDa (as indicated).

kDa SH-SY5Y HT-29

206

DCC

118

NGL-3/LRRC4B in Human Cerebellum. Netrin-G Ligand 3 (NGL-3)/LRRC4B was Detection of Human DCC by Western Blot. Western blot shows lysates of Phospho-ERK1/ERK2 in Rat Cortex. Phosphorylated ERK1/ERK2 was detected in detected in immersion-fi xed paraffi n-embedded sections of human cerebellum the SH-SY5Y human neuroblastoma cell line and the HT-29 human colon perfusion-fi xed frozen sections of rat brain using a Rabbit Anti-Human/Mouse/Rat using a Mouse Anti-Human NGL-3/LRRC4B Monoclonal Antibody (Catalog # adenocarcinoma cell line. The PVDF membrane was probed with a Sheep Anti- Phospho-ERK1/ERK2 (ERK1 T202/Y204, ERK2 T185/Y187) Antigen Affi nity-purifi ed MAB6919). Before incubation with the primary antibody, the tissue was subjected Human DCC Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5884) Polyclonal Antibody (Catalog # AF1018). The tissue was stained with the Anti-Rabbit to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog followed by a HRP-conjugated Donkey Anti-Sheep IgG Secondary Antibody (Catalog HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS005; brown) and counterstained # CTS013). The tissue was stained using the Anti-Mouse HRP-DAB Cell & Tissue # HAF016). DCC was detected at approximately 180 kDa (as indicated). with hematoxylin (blue). Staining Kit (Catalog # CTS002; brown) and counterstained with hematoxylin (blue). Specifi c staining was localized to the cytoplasm and processes of Purkinje neurons.

www.RnDSystems.com/go/Netrins 15 NEURAL DEVELOPMENT & AXON GUIDANCE

Semaphorins, Plexins, & Related Molecules Semaphorins (Sema) are an evolutionarily conserved family of molecules governing attractive Sema3E signaling also contains VEGF R2.6 These that aff ect axon guidance, cell migration, synaptogenesis, and dendritic studies determined that Plexin D1 and VEGF R2 acted as the ligand- spine formation.1 Divided into eight subfamilies, semaphorins are binding and signal transduction subunits, respectively. detected in invertebrates (Sema1 and 2), vertebrates (Sema3-7), and Transmembrane Sema4s generally bind to class B and C1 Plexin receptors, viruses (Sema8). All semaphorins are characterized by an extracellular  and Plexin B-ErbB2 interactions are required for some Sema4D functions.7 propeller-structured N-terminal semaphorin (sema) domain, and with the Sema5s act as attractants through heparin sulfate proteoglycans (HSPGs), exception of viral semaphorins, also contain a plexin-semaphorin-integrin as repellents through chondroitin sulfate proteoglycans (CSPGs), (PSI) domain.2,3 Members of this diverse group of signaling molecules can and also bind Plexin B receptors. Plexin A receptors are targeted by be secreted (Sema2, 3, 8), transmembrane (Sema1, 4-6), or glycosylphos- transmembrane Sema6 molecules. Transmembrane Semaphorins may phatidylinositol (GPI)-linked to the plasma membrane (Sema7). Originally also serve as recep tors to induce reverse signaling in the host cell.8 In described as chemorepellents, semaphorins are now considered bi- addition, transmembrane Semaphorins may exert eff ects in the host cell functional, in that they can act as attractants or repellents, depending on by interacting in cis with Plexin receptors on the same plasma membrane.9 the biological context in which signaling occurs. Members of the GPI-linked Sema7 subfamily bind to Plexin C1 and also Semaphorin signaling is dependent on a variety of receptor molecules, function through neuronal Integrin receptors. Finally, Sema8s are viral the most prominent of which are the plexin subfamilies (A-D).4 Plexins are mimics of Sema7s and function through Plexin C1 receptors. type I transmembrane proteins that interact with semaphorins through an References extracellular sema domain. These proteins function as signaling receptors 1. Pasterkamp, R.J. & Giger, R.J. (2009) Curr. Opin. Neurobiol. 19:263. for multiple semaphorin subfamilies. Transduction of semaphorin signals often requires the presence of co-receptors. For example, invertebrate 2. Koppel, A.M. et al. (1997) Neuron 19:531. Sema1 binds to Plexin A, which interacts with the co-receptor Off -track 3. Liu, H. et al. (2010) Cell 142:749. (OTK). Sema2 signals through Plexin B, which may form heteromultimeric 4. Jackson, R.E. & Eickholt, B.J. (2009) Curr. Biol. 19:R504. receptor complexes with Plexin A. 5. Derijck, A.A. et al. (2010) Trends Cell Biol. 20:568.

With the exception of Sema3E, Sema3s signal through class A Plexin 6. Bellon, A. et al. (2010) Neuron 66:205. receptors following binding to a Neuropilin (NRP)-1 or -2 co-receptor.5 7. Swierzc, J.M. et al. (2004) J. Cell. Biol. 165:869. Immunoglobulin (Ig)-superfamily cell adhesion molecules (IgCAMs) also serve as additional co-receptors for Sema3A-D, F, and G. In contrast, 8. Yu, L. et al. (2010) J. Neurosci. 30:12151. Sema3E binds directly to Plexin D1, and the additional presence of NRP- 9. Haklai-Topper, L. et al. (2010) EMBO J. 29:2635. 1 converts Sema3E from a repellent to an attractant. Recent studies of axon tract formation in mouse brain suggest that the receptor complex

Invertebrate Vertebrate Viral Sema1 Sema2 Sema3A-D,F, G Sema3E Plexin B Sema4 Sema4D Sema5 Sema6Plexin A Sema7 Sema8

cis cis

CSPG HSPG NRP IgCAM NRP-1 Integrin OTK Plexin A Plexin B Plexin A Plexin D1 VEGF R2 ErbB2 Plexin B Plexin A Plexin C1 Plexin C1 Plexin B Plexin B

Domain Key: Ig-like Sema PSI IPT Split GAP Convertase cleavage Tyrosine kinase PDZ-binding Basic Meprin GTPase-binding Complement-binding Coagulation Factor V/VIII Ligand-binding Thrombospondin-like GPI anchor

Figure 12. Semaphorin Classiﬁ cation, Domain Structure, and Receptor Interactions. Semaphorins signal via specifi c receptors and are dependent on a variety of coreceptor molecules. Please see main text for full description of ligand-receptor relationships.

16 For research use only. Not for use in diagnostic procedures. NEURAL DEVELOPMENT & AXON GUIDANCE

R&D Systems Products for Semaphorins, Plexins, & Related Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs Semaphorins Plexins

Semaphorin 3A H M H (FC, WB) M (FC) Plexin A1 MH (IHC, WB) M (FC, IHC, WB)

Semaphorin 3B M Plexin A2 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB)

Semaphorin 3C H M M (IHC, WB) Plexin A3 M (IHC, WB) R (IHC, WB)

Semaphorin 3E H M H (FC, IHC, WB) M (IHC, WB) H Plexin A4 HH (FC, WB) M (WB) R (WB)

Semaphorin 3F MH (WB) M (WB) Plexin B1 H (FC, IHC, WB)

Semaphorin 4A H Plexin B2 H M H (FC, IHC, WB)

Semaphorin 4B H (FC, WB) M (WB) Plexin B3 HH (WB) M (FC, IHC, WB) R (FC, IHC, WB)

Semaphorin 4C H (IHC, WB) M (IHC, WB) Plexin C1 H M H (FC, IHC, WB) M (FC, IHC, WB)

Semaphorin 4D/ MM (FC, IHC, WB) Plexin D1 HH (FC, WB) CD100 TEM7/PLXDC1 H (WB) Semaphorin 4G HH (IHC, WB) M (WB) Neuropilins Semaphorin 5A H M H (IHC, WB) M (IHC, WB) R (IHC, WB) Neuropilin-1 H M R H (B/N, FC, IHC, WB) M (FC) R (B/N, FC, IHC, WB) Semaphorin 5B H M Neuropilin-2 H R H (B/N, FC, IHC, WB) R (B/N, FC, WB) Semaphorin 6A HH (FC, IHC, WB) M (IHC, WB) Other Semaphorin Related Molecules Semaphorin 6B HH (IHC, WB) M (WB) CD72 H (WB) M (IHC, WB) Semaphorin 6C H M H (WB) M (IHC, WB) ErbB2/Her2 HH (B/N, ELISA, FC, IHC, WB) M (FC, IHC, WB) H Semaphorin 6D H (FC, WB) TIM-2 MM (WB) Semaphorin 7A H M H (FC, WB) M (WB) VEGF R2/KDR/Flk-1 H M H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M

A. B.

kDa Mouse Brain A. B. 199 Plexin A2

kDa EmbryoRat Hippocampal Neuron 117

80 118 Plexin B3 97 52

37 54

37 29

Semaphorin 3A-induced Growth Cone Collapse. A fully extended chick dorsal Detection of Mouse Plexin A2 and Human Plexin B3 by Western Blot. Western Plexin B2 in Mouse Choroid Plexus. Plexin B2 was detected in perfusion-fi xed root ganglion growth cone grown in the presence of Recombinant Human -NGF blots show lysates of mouse brain tissue and rat embryonic hippocampal neurons. frozen sections of mouse choroid plexus using a Sheep Anti-Human Plexin B2 (Catalog # 256-GF) was untreated (A) or treated with Recombinant Human A. The PVDF membrane was probed with a Rat Anti-Mouse Plexin A2 Monoclonal Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5329). The tissue Semaphorin 3A (Catalog # 1250-S3; B). Treatment with Recombinant Human Antibody (Catalog # MAB5486) followed by a HRP-conjugated Goat Anti-Rat IgG was stained with the NorthernLights™ 557-conjugated Donkey Anti-Sheep IgG Semaphorin 3A induced growth cone collapse. Secondary Antibody (Catalog # HAF005). B. The PVDF membrane was probed Secondary Antibody (Catalog # NL010; red) and counterstained with DAPI (blue). with a Sheep Anti-Human Plexin B3 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF4958) followed by a HRP-conjugated Donkey Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). Plexin A2 and Plexin B3 were detected at approximately 196 kDa and 140 kDa, respectively (as indicated).

www.RnDSystems.com/go/Semaphorins 17 NEURAL DEVELOPMENT & AXON GUIDANCE

Slit Ligands & ROBO Receptors Originally discovered in Drosophila, Slit proteins bind to Roundabout Studies using Drosophila identifi ed ROBO1 as a critical factor controlling (ROBO) receptors to infl uence axon guidance, cell migration, cell midline crossing. In Drosophila, transient high levels of Commissureless adhesion, and angiogenesis.1 Slit ligands are secreted glycoproteins that at the midline direct ROBO1 receptors to the endosome for degradation, also interact with components of the extracellular matrix (ECM). The three thus allowing midline crossing.5 In contrast, diff erential gene expression mammalian homologues (Slit1-3) are expressed throughout the central of ROBO2 and ROBO3 was shown to determine the lateral positioning of nervous system and in most organs. Structurally, Slit proteins contain longitudinal axons following midline crossing.6 four leucine-rich repeats, seven to nine EGF-like sequences, a laminin-G In mammalian studies, ROBO3, which is exclusively expressed in domain, and a C-terminal cysteine-rich domain. commissural axons, was shown to govern midline crossing (Figure 13). Vertebrate neurons express three ROBO receptors (ROBO1-3), which Although the ability of ROBO3 to bind Slit is unconfi rmed, alternative contain fi ve immunoglobulin (Ig)-like domains and three fi bronectin type splicing results in two isoforms that exert opposite actions. Recent studies III repeats in the extracellular region. The second leucine-rich domain of suggest that in pre-crossing axons, ROBO3.1 expression suppresses the the Slit proteins binds to the fi rst two Ig-like domains of ROBO receptors. repulsive eff ects of Slit-ROBO1/2 to permit midline crossing.7 In post- The intracellular portion of the molecule includes four (ROBO1/2) or crossing axons, the expression of ROBO3.2 cooperates with ROBO1/2 to three (ROBO3) conserved cytoplasmic domains. A fourth ROBO receptor repel projections away from the fl oor plate. Furthermore, in the presence (ROBO4) is expressed in endothelial cells and is believed to be involved in of Slit, ROBO1 has been shown to bind the Netrin receptor DCC and silence angiogenesis.2 Heparan sulfate proteoglycans (HSPGs) are very important Netrin mediated attraction.8 Additional studies indicate that cleavage of for Slit/ROBO signaling. HSPGs potentiate Slit activity by stabilizing the ROBO by metalloproteinases may also be an important factor mediating interaction between Slit and ROBO.3 In addition, HSPGs bind to other axon repulsion at the midline.9 components of the ECM to facilitate cell adhesion. References Slits are established repulsive factors in the guidance of commissural 1. Ypsilanti, A.R. et al. (2010) Development 137:1939. axons. Commissural axons project across the midline of the spinal cord 2. Marlow, R. et al. (2010) Proc. Natl. Acad. Sci. USA 107:10520. and contact target cells on the opposite side of the CNS. Current research 3. Seiradake, E. et al. (2009) EMBO Rep. 10:736. suggests that commissural axons are attracted towards the fl oor plate 4. Dickson, B.J. & Y. Zou (2010) Cold Spring Harb. Perspect. Biol. 2:a002055. by increasing concentrations of Netrin-1 and Sonic Hedgehog (Shh). After crossing the midline, axons are repelled away from the fl oor plate, 5. Keleman, K. et al. (2005) Nat. Neurosci. 8:156. and prevented from recrossing the midline by Slits and Semaphorins. 6. Spitzweck, B. et al. (2010) Cell 140:409. Critically, these sequential events require pre-crossing axons to ignore 7. Jaworski, A. et al. (2010) J. Neurosci. 30:9445. midline repellents and post-crossing axons to be insensitive to midline 8. Stein, E. & M. Tessier-Lavinge (2001) Science 291:1928. attractants. How this is achieved has been the subject of intense study.4 9. Coleman, H.A. et al. (2010) Development 137:2417.

Slit Slit Slit

Netrin-1 Slit DCC Homodimer DCC Homodimer HSPG HSPG

Netrin Netrin

n ROBO3.2 ROBO1/2 ROBO3.1 o ROBO1/2 ROBO1 Growth cone

p q r

p Commissural axon qr no Floor plate ATTRACTION REPULSION REPULSION ATTRACTION

Domain Key: Ig-like Fibronectin type-III Conserved IC Heparin-binding Cysteine-rich EGF repeat Leucine-rich Laminin-like

Figure 13. Commissural Axon Guidance Across the Midline. Commissural axons are guided ventrally from the dorsal spinal cord by increasing concentrations of Netrin-1, which transduces signals through its receptor DCC . In parallel, growth cones are repelled by Slit binding to ROBO1/2 . In proximity to the fl oor plate, ROBO3.1 inhibits ROBO1/2-induced repulsion to allow midline crossing . After crossing the midline, ROBO3.2 cooperates with ROBO1/2 to repel axons away from the fl oor plate . In addition, ROBO1 binds DCC to silence Netrin-1-mediated attraction .

18 For research use only. Not for use in diagnostic procedures. NEURAL DEVELOPMENT & AXON GUIDANCE

R&D Systems Products for Slit & ROBO Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs ROBO Receptors Slit Ligands

ROBO1 RH (WB) R (ELISA, IHC, WB) R Slit1 H M

ROBO2 HH (FC, IHC, WB) Slit2 M

ROBO3 H M H (IHC, WB) M (IHC, WB) Slit3 MH (B/N, WB) M (B/N, WB)

ROBO4 HH (FC, WB) M (FC)

A. B.

kDa SH-SY5Y A. B. kDa Neuro-2A

210 ROBO3 206

DCC

118

117 97

Slit2 Enhances Neurite Outgrowth. Cultured chick dorsal root ganglion neurons Detection of Mouse ROBO3 and Human DCC by Western Blot. Western blot ROBO1 in Rat Neural Tube. Roundabout Receptor 1 (ROBO1) was detected in were grown in the presence of Recombinant Human -NGF (Catalog # 256-GF; shows lysates of the Neuro-2A mouse neuroblastoma cell line and the SH-SY5Y immersion-fi xed frozen sections of rat embryo using a Goat Anti-Rat ROBO1 Antigen 50 ng/mL), on plates coated with PBS (A) or on plates coated with 12 g/mL human neuroblastoma cell line. A. The PVDF membrane was probed with a Goat Affi nity-purifi ed Polyclonal Antibody (Catalog # AF1749). The tissue was stained Recombinant Mouse Slit2 (Catalog # 5444-SL; B). The presence of Recombinant Anti-Mouse ROBO3 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS008; brown) Mouse Slit2 signifi cantly enhanced neurite outgrowth. AF3155) followed by a HRP-conjugated Chicken Anti-Goat IgG Secondary Antibody and counterstained with hematoxylin (blue) (Catalog # HAF019). B. The PVDF membrane was probed with a Sheep Anti-Human DCC Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5884) followed by a HRP-conjugated Donkey Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). ROBO3 and DCC were detected at approximately 205 kDa and 180 kDa, respectively (as indicated).

SH-SY5Y 70 kDa 60 200 ROBO1 50

40 116 30 80

Relative Cell Number Cell Relative 20

53 10

0 37 100 101 102 103 104 ROBO4

Detection of Human ROBO1 by Western Blot. Western blot shows lysates of the Detection of ROBO4 by Flow Cytometry. Human umbilical vein endothelial cells ROBO3 in Mouse Embryo. Roundabout Receptor 3 (ROBO3) was detected in SH-SY5Y human neuroblastoma cell line. The PVDF membrane was probed with a were stained with a Mouse Anti-Human ROBO4 Monoclonal Antibody (Catalog # immersion-fi xed frozen sections of mouse embryo (E11.5) using a Goat Anti-Mouse

Sheep Anti-Human ROBO1 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # MAB24541, fi lled histogram) or a Mouse IgG2B Isotype Control (Catalog # MAB0041, ROBO3 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF3155). The tissue AF7118) followed by a HRP-conjugated Donkey Anti-Sheep IgG Secondary Antibody open histogram), followed by an APC-conjugated Anti-Mouse IgG Secondary was stained using the NorthernLights™ 557-conjugated Donkey Anti-Goat IgG (Catalog # HAF016). ROBO1 was detected at approximately 200 kDa (as indicated). Antibody (Catalog # F0101B). Secondary Antibody (Catalog # NL001; red) and counterstained with DAPI (blue). Specifi c staining was localized to developing forebrain.

www.RnDSystems.com/go/ROBO 19 NEURAL DEVELOPMENT & AXON GUIDANCE

Wnt Ligands, Frizzled Receptors, & Related Molecules Wingless/Int ligands (Wnts) are a family of secreted glycoproteins that suggest that Wnt and Sonic Hedgehog (Shh) cooperate to guide post- exert critical eff ects throughout embryogenesis. During development, crossing axons by establishing a Shh-dependent gradient of sFRP1.8 Wnt signaling controls cell proliferation and migration, stem cell fate, Experiments using cerebellar mossy fi bers found that Wnt-7a increased and the establishment of polarity and tissue patterning.1 Wnt ligands Synapsin I clustering and growth cone enlargement, suggesting Wnt induce their eff ects by binding to the Frizzled family of G protein-coupled ligands also infl uence synapse formation.9 In support of this hypothesis, receptors to activate one of three signaling pathways.2,3 Canonical Wnt Wnt-3a was shown to promote spinal sensory neuron growth cone signaling is dependent on the stabilization and nuclear translocation of remodeling, a process that precedes synaptogenesis.10 At presynaptic -Catenin. In the absence of Wnt, -Catenin is phosphorylated by GSK-3 membranes, Wnt-7a induces 7-nicotinic acetylcholine receptor traf- and degraded via ubiquitin-mediated proteasomal degradation. However, fi cking, synaptic vesicle recycling, and the recruitment of Frizzled-5.11-13 following Wnt binding to a Frizzled receptor, -Catenin translocates to the  Postsynaptic actions of Wnt-5a include induction of GABA-A receptor nucleus where it complexes with TCF/LEF transcription factors to activate expression, dendritic spine morphogenesis, and insertion of NMDA target genes. Alternatively, Wnt-Frizzled interactions can induce the Wnt- receptors.14-16 calcium pathway, which is dependent on the stimulation of calcium- responsive second messenger systems, or the planar cell polarity (PCP) Wnt ligands are also believed to be important modulators of neurogenesis. pathway, which requires the activation of Rho-type GTPases and Jun For instance, Wnt-7a was shown to promote the maturation of neural 17 N-terminal serine/threonine kinases (JNK). precursors to neurons in the developing mouse neocortex. In adult brain, Wnt-3 is thought to be an intrinsic regulator of neurogenesis in Currently, there are nineteen known Wnt ligands and ten identifi ed Frizzled the dentate gyrus of the hippocampus (Figure 14).18 Because Wnt ligands receptors. In addition, lipoprotein receptor-related proteins LRP5 and exert positive actions on neurogenesis and synaptogenesis, studies have LRP6 complex with Frizzled receptors to modulate canonical signaling. investigated the potential use of these proteins to combat the progression Recent studies also revealed that Wnt ligands bind to ROR1, ROR2, and Ryk of Parkinson’s disease (PD) and Alzheimer’s disease (AD). PD-related receptor tyrosine kinases to aff ect non-canonical signaling.4,5 In addition, studies have focused on the generation of dopaminergic neurons from Wnt signaling is attenuated by secreted inhibitors, such as Wnt inhibitory midline progenitor cells, a process that is promoted by the release of Wnt- factor (WIF-1) and secreted Frizzled-related proteins (sFRP). 1.19 In parallel, Wnt-5a and Wnt-3a have been shown to prevent synaptic The complexity of Wnt biology is underlined by the fact that the same damage induced by oligomeric forms of the AD toxin amyloid-(A).16,20 Wnt ligand can exert opposing eff ects depending on the environmental References context in which signaling occurs. For example, commissural axon 1. van Amerongen, R. & R. Nusse (2010) Development 136:3205. guidance studies determined that Wnt-1 and Wnt-5a act as chemo- 2. Freese, J.L. et al. (2010) Neurobiol. Dis. 38:148. attractants through Frizzled receptors and as repellents via Ryk.6,7 In addition, a gradient of Wnt-4 is believed to attract anterior-directed 3. Inestrosa, N.C. & E. Arenas (2010) Nat. Rev. Neurosci. 11:77. projections after midline crossing via Frizzled-3.7 More recent studies 4. Paganoni, S. et al. (2010) Neuroscience 165:1261. 5. Fradkin, L.G. et al. (2010) Trends Neurosci. 33:84.

6. Liu, Y. et al. (2005) Nat. Neurosci. 8:1151.

from Entorhinal Cortex 7. Lyuksyutova, A.I. et al. (2003) Science 302:1984. 8. Domanitskaya, E. et al. (2010) J. Neurosci. 30:11167. Granule Layer 9. Hall, A.C. et al. (2000) Cell 100:525.

Subgranular Zone 10. Purro, S.A. et al. (2008) J. Neurosci. 28:8644.

to CA3 Differentiating Granule Neuron 11. Sahores, M. et al. (2010) Development 137:2215.

E-Catenin 12. Farias, G. et al. (2007) J. Neurosci. 27:5313. TCF/LEF NeuroD1 13. Cerpa, W. et al. (2008) J. Biol. Chem. 283:5918. Neural Stem Cell HDAC1 14. Cuitino, L. et al. (2010) J. Neurosci. 30:8411. SOX2 E-Catenin NeuroD1 TCF/LEF LINE-1 retrotransposon 15. Varela-Nallar, L. et al. (2010) Proc. Natl. Acad. Sci. USA 107:21164. HDAC1 SOX2 LINE-1 16. Cerpa, W. et al. (2010) Mol. Neurodegen. 5:3. retrotransposon SOX/LEF element 17. Hirabayashi, Y. et al. (2004) Development 131:2791. Wnt-3a 18. Kuwubara, T. et al. (2009) Nat. Neurosci. 12:1097.

19. L’episcopo, F. et al. (2011) Neurobiol. Dis. 41:508. Astrocyte 20. Shruster, A. et al. (2011) J. Neurochem. 116:522.

Figure 14. Sox/LEF Elements Regulate NeuroD1 Expression and Neurogenesis in the Adult Hippocampus. Neural stem cells in the subgranular zone of the dentate gyrus diff erentiate toward mature granule neurons following the release of Wnt-3a from astrocytes.18 Recent data suggest that diff erentiation is dependent on a Sox/LEF dual site within the NeuroD1 promoter sequence. Following eleaser of Wnt-3a, a Sox2/HDAC1 repressor complex is replaced by a -Catenin/TCF/LEF activation complex, which leads to NeuroD1 expression and neural diff erentiation. Binding of -Catenin/TCF/LEF also promotes the expression of LINE-1, a retrotransposon thought to be important for neuronal diversifi cation.

20 For research use only. Not for use in diagnostic procedures. NEURAL DEVELOPMENT & AXON GUIDANCE

R&D Systems Products for Wnt Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs Wnt Ligands Wnt Receptors

Wnt-1 M (IHC, WB) CELSR2 H (IHC, WB)

Wnt-2 H (IHC, WB) Frizzled-1 H M H (FC, IHC, WB) M (FC, IHC, WB)

Wnt-2b M (IHC, WB) Frizzled-2 MM (WB)

Wnt-3a H M H (B/N, WB) M (B/N, WB) Frizzled-3 H (FC, IHC, WB) M (FC, IHC, WB)

Wnt-4 H M H (WB) M (IHC, WB) Frizzled-4 H M H (FC, IHC, WB) M (FC, IHC, WB)

Wnt-5a H M H (IHC, WB) M (IHC, WB) R (IHC, WB) Frizzled-5 HH (WB)

Wnt-5b MM (IHC) Frizzled-6 H (FC, WB) M (FC, IHC, WB)

Wnt-6 H (IHC, WB) Frizzled-7 H M H (FC, IHC) M (FC, IHC, WB)

Wnt-7a HH (IHC, WB) Frizzled-8 H M M (IHC, WB)

Wnt-7b H (IHC, WB) Frizzled-9 M (IHC, WB)

Wnt-8a M (IHC, WB) Frizzled-10 H

Wnt-8b H (IHC, WB) M (IHC, WB) LRP-1 H (FC, WB)

Wnt-9a H (IHC, WB) LRP-1 Cluster II HH (WB)

Wnt-9b MH (WB) M (IHC, WB) LRP-1 Cluster III HH (IHC, WB)

Wnt-10b MM (WB) LRP-1 Cluster IV H

Wnt-11 HH (IHC, WB) M (IHC, WB) LRP-1B H (WB)

Species Key: H Human M Mouse R Rat LRP-4 H (IHC, WB) R (IHC, WB) Application Key: B/N Blocking/Neutralization ELISA ELISA Capture and/or Detection FC Flow Cytometry IHC Immunohistochemistry WB Western blot LRP-6 H M H (FC, IHC, WB) M (WB)

ROR1 Receptor H (FC, WB) H Tyrosine Kinase

RTK-like Orphan H (FC, WB) H Receptor 2/ROR2

Ryk H (WB) M (IHC, WB) mbryo E d.p.c.) A. B. A. B. Mouse kDa (13 204 _

117 _ kDa Human Hypothalamus 78 _

52 _ 53 Wnt-11 37 _ 37 Wnt-2 29 _ 29 20 _ 19 7 _

Wnt-1 in Mouse Embryonic Spinal Cord. Wnt-1 was detected in immersion-fi xed Detection of Human Wnt-2 and Mouse Wnt-11 by Western Blot. Western blot Wnt-induced Stress Fiber Formation and Nuclear -Catenin Accumulation. frozen sections of mouse embryonic spinal cord (13 d.p.c.) using a Goat Anti-Mouse shows lysates of human hypothalamus and mouse embryo (13 d.p.c.) tissue. A. NIH-3T3 mouse embryonic fi broblast cells were untreated (A) or treated with Wnt-1 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF1620). The tissue The PVDF membrane was probed with a Goat Anti-Human Wnt-2 Antigen Affi nity- Recombinant Mouse Wnt-3a (Catalog # 1324-WN; B). Treatment with Recombinant was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (Catalog # purifi ed Polyclonal Antibody (Catalog # AF3464) followed by a HRP-conjugated Mouse Wnt-3a promoted stress fi ber formation (red) and nuclear -Catenin CTS008; brown) and counterstained with hematoxylin Chicken Anti-Goat IgG Secondary Antibody (Catalog # HAF019). B. The PVDF accumulation (green). Please visit our website for information about our new high membrane was probed with a Rat Anti-Mouse Wnt-11 Monoclonal Antibody purity human Wnt-3a (Catalog # 5036-WNP). Images Courtesy of Dr. Raymond Habas, (Catalog # MAB3746) followed by a HRP-conjugated Goat Anti-Rat IgG Secondary Robert Wood Johnson School of Medicine, Piscataway, NJ. Antibody (Catalog # HAF005). Wnt-2 and Wnt-11 were detected at approximately 40 kDa and 50 kDa, respectively (as indicated).

www.RnDSystems.com/go/Wnt 21 NEURAL DEVELOPMENT & AXON GUIDANCE

R&D Systems Products for Wnt Family Molecules RECOMBINANT RECOMBINANT MOLECULE ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs PROTEINS Wnt Inhibitors Wnt Pathway Modulators

Dkk-1 H M R H (B/N, ELISA, IHC, WB) M (ELISA, IHC, WB) R (WB) H M Axin-2 H (IHC)

Dkk-2 H M M (IHC, WB) DISC1 H (IHC, WB)

Dkk-3 HH (ELISA, IHC, WB) M (WB) H Glypican 1 H M H (FC, IHC, WB)

Dkk-4 H M H (ELISA, WB) M (WB) H Glypican 3 H M H (FC, IHC, WB)

sFRP-1 HH (WB) Glypican 5 H M H (FC, WB) M (FC, WB)

sFRP-2 MM (WB) Kremen-1 MH (IHC, WB) M (ELISA, FC, IHC, WB) M

sFRP-3 H M H (ELISA, WB) M (ELISA, IHC, WB) H Kremen-2 H M R H (FC, WB) M (WB)

sFRP-4 HH (WB) LRP-5 H (WB) sFRP-5 H MESDC2 MH (IHC, WB) M (IHC, WB)

Soggy-1/DkkL1 H M H (WB) , WB) M MFRP HH (IHC, WB) M (IHC, WB)

WIF-1 HH (ELISA, IHC, WB) M (WB) H Myocilin H (WB)

Species Key: H Human M Mouse R Rat NeuroD1 H (IHC, WB) M (IHC, WB) Application Key: B/N Blocking/Neutralization ELISA ELISA Capture and/or Detection FC Flow Cytometry IHC Immunohistochemistry WB Western blot Norrin H M H (B/N, IHC, WB) M (WB) Nucleoredoxin H (WB)

R-Spondin 1 H M H (B/N, WB) M (WB)

R-Spondin 2 H M H (WB)

R-Spondin 3 H M H (B/N, WB) M (WB)

R-Spondin 4 H M H (WB) M (B/N, WB)

Shisa-4 H (WB) M (WB)

SOST/Sclerostin H M H (ELISA, WB) M (IHC, WB) H

Wnt-3a Antibody ( g/mL) P 50 0.01 0.1 0 10 100 kDa Huh-7 C6 NIH-3T3 1.6 1.6 Alkaline PhosphataseAlkaline (Mean O.D.) 100 40 β-Catenin 1.2 1.2 80 30 60 50 0.8 0.8 20 40 Relative Cell Number Cell Relative 10 0.4 0.4 30 Alkaline Phosphatase (Mean O.D.) Alkaline Phosphatase 0.0 0.0 0 0.01 0.1 1 10 100 101 102 103 104 Wnt-3a (ng/mL) 20 E-Catenin

Alkaline Phosphatase Production Induced by Wnt-3a and Neutralization by Detection of Human/Mouse/Rat -Catenin by Western Blot. Western blot shows Detection of -Catenin by Flow Cytometry. The HeLa human cervical epithelial a Wnt-3a Antibody. Recombinant Mouse Wnt-3a (Catalog # 1324-WN) induces lysates of the Huh-7 human hepatoma cell line, the C6 rat glioma cell line, and the carcinoma cell line was stained with a Mouse Anti-Human/Mouse/Rat -Catenin alkaline phosphatase production in the MC3T3-E1 mouse preosteoblast cell line in a NIH-3T3 mouse embryonic fi broblast cell line. The PVDF membrane was probed Monoclonal Antibody (Catalog # MAB13292, fi lled histogram) or a Mouse IgG2A dose-dependent manner (green line). Under these conditions, alkaline phosphatase with a Mouse Anti-Human/Mouse/Rat -Catenin Monoclonal Antibody (Catalog # Isotype Control (Catalog # MAB003, open histogram), followed by an APC- production elicited by Recombinant Mouse Wnt-3a (10 ng/mL) is neutralized (gray MAB1329) followed by a HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody conjugated Goat Anti-Mouse IgG Secondary Antibody (Catalog # F0101B). line) by increasing concentrations of a Rat Anti-Human/Mouse Wnt-3a Monoclonal (Catalog # HAF007). -Catenin was detected at approximately 95 kDa (as indicated). Antibody (Catalog # MAB1324).

22 For research use only. Not for use in diagnostic procedures. NEURAL DEVELOPMENT & AXON GUIDANCE

R&D Systems Products for Wnt Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs Canonical Wnt Intracellular Signaling Non-canonical Wnt Intracellular Signaling

APC H (IHC, WB) CaM Kinase II H (WB) M (WB) R (WB) X (WB)

ASCL1/Mash1 M (IHC, WB) Casein Kinase 1α H (WB) M (WB) R (WB)

ASCL2/Mash2 H (IHC, WB) Casein Kinase 1γ H (WB) M (WB) R (WB)

Axin-1 H (WB) M (WB) R (WB) Casein Kinase 1δ H (WB) M (WB) R (WB)

Axin-2 H (IHC) Casein Kinase 1ε H (WB) M (WB) R (WB)

β-Catenin H (ChIP, FC, IHC, WB) M (ChIP, FC, IHC, WB) H Casein Kinase 2β H (WB) R (ChIP, FC, IHC, WB) X (WB) Dishevelled-1 H (IHC, WB) Bcl-9 H (WB) Dishevelled-2 H (WB) Bcl9-2 H (WB) Dishevelled-3 H (IHC, WB) c-Abl H (WB) JNK H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R Ccd1/DIXDC1 M (IHC, WB) JNK1/JNK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) CREB H (ChIP, IHC, WB) M (WB) R (WB) H M R JNK1 MH (IHC, WB) M (IHC, WB) R (IHC, WB) Dishevelled-1 H (IHC, WB) JNK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R Dishevelled-2 H (WB) JunB H (WB) Dishevelled-3 H (IHC, WB) c-Jun H (IHC, WB) M (IHC, WB) Draxin H M H (IHC, WB) M (IHC, WB) R (IHC, WB) JunD H (WB) M (WB) GSK-3α H (IHC, WB) M (IHC, WB) R (IHC, WB) H MKK7 H (IHC, WB) GSK-3α/β H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R PKCα H (WB) M (WB) R (WB) GSK-3β HH (FC, IHC, WB) M (FC, WB) R (FC, WB) H M R PKCβ1 H (IHC, WB) R (IHC, WB) Pygopus-1 H (WB) M (WB) PKCβ2 H (WB) M (WB) Pygopus-2 H (IHC, WB) PKCγ H (WB) M (WB) R (WB) TCF7/TCF1 H (IHC, WB) PKCε H (IHC, WB) M (IHC, WB) R (IHC, WB) TCF7L1/TCF3 H (IHC, WB) M (WB) PKCδ HH (WB) Ubiquitin H Ms Pl Pz Y H (IHC, WB) H Rb Ms PKCι/λ H (WB) M (WB) Ubiquitin/ H (IHC, WB) Ubiquitin+1 PKCι/λ/ζ H (IHC, WB) M (IHC, WB) R (IHC, WB)

Ubiquitin+1 HH (ELISA, IHC, WB) H PKCν H (WB) Di-Ubiquitin/Ub2 H Ms Ms PKCθ H (FC, IHC, WB) M (WB) Tri-Ubiquitin/Ub3 H PKCζ H (WB) Tetra-Ubiquitin/Ub4 H ROCK1 HH (WB) M (WB) R (WB) Penta-Ubiquitin/Ub5 H ROCK2 H (WB) M (WB) R (WB) Hexa-Ubiquitin/Ub6 H TAK1 H (WB) Octa-Ubiquitin/Ub8 H Vang-like Protein 1/ H (IHC, WB) VANGL1 Poly-Ubiquitin H Vang-like Protein 2/ H (IHC, WB) M (IHC, WB) R (IHC, WB) VANGL2

www.RnDSystems.com/go/Wnt 23 NEURAL DEVELOPMENT & AXON GUIDANCE

Nogo Proteins & Receptors Discovered as molecules that block axon regeneration following injury, Nogo coupled receptor 50 (GPR50) has been identifi ed as a possible candidate.8 proteins are one of the most potent neurite growth inhibitors in the CNS.1 Collectively, these data suggest that Nogo-A binds to a multi-unit receptor Members of the reticulon (RTN) family, Nogo proteins function as negative complex, in a manner similar to Neurotrophins, Netrins, and Semaphorins. regulators during development and serve as stabilizers of neuronal wiring In addition, variable membrane topologies, diff er ential subcellular in adult brain.2 Alternative splicing and diff erential promoter usage results in expression, and multiple signal transduction systems permit a diverse the generation of three protein products (Nogo-A, -B, and -C) from a single range of functions in the developing and adult CNS. gene (RTN4/NOGO). The RTN domain contains two hydrophobic stretches Nogo-A is believed to exert its inhibitory eff ects by modulating the that can each span the cell or endoplasmic reticulum (ER) membrane equilibrium of Rac1/RhoA eff ector kinases, which regulate F-Actin dyna mics twice and are linked together by a sixty-six amino acid sequence termed in the growth cone. Both the Nogo-66 and Nogo-20 domains have been Nogo-66. In addition, Nogo proteins lack a signal sequence, permitting the shown to eff ect the small GTPases Rac1 and RhoA, which induce antagonis- adoption of several diff erent membrane topologies. Expressed at the ER tic eff ects on neurites.9 Rac1 activates PAK1 to promote growth cone motility, and plasma membranes, alternate topologies result in luminal/extracellular whereas RhoA stimulates ROCK to induce growth cone collapse. Recent or intracellular localization of the N-terminal domain. studies suggest that one mechanism underlying the actions of Nogo requires The inhibitory actions of Nogo-A are dependent on two domains, Nogo- internalization of the tripartite receptor complex via Pincher-dependent 66, which is present in all three Nogo isoforms, and a sequence referred to macroendocytosis.10 These studies indicated that ligand-receptor complexes as Nogo-20, which is unique to the long N-terminal domain of Nogo-A.3 are transported to the cell body in signaling endosomes (signalosomes), Nogo-A exerts its eff ects by binding to Nogo Receptor 1 (NgR1) via the where they cause changes in the transcriptional machinery (Figure 15B). Nogo-66 domain. Because NgR1 is glycosylphosphatidylinositol (GPI)- References linked to the plasma membrane, signal transduction requires the form- 1. Caroni, P. et al. (1988) Neuron 1:85. ation of a tripartite receptor complex that includes neurotrophin receptor 2. Schwab, M. (2010) Nat. Rev. Neurosci. 11:799. p75 (NGF R) and the leucine-rich repeat and Ig domain containing Nogo receptor-interacting protein-1 (LINGO-1) (Figure 15A).4 When localized to 3. Oertle, T. et al. (2003) J. Neurosci. 23:5393. the plasma membrane, LINGO-1 can be replaced as the signal transducer 4. Mi, S. et al. (2004) Nat. Neurosci. 7:221. 5 by TROY, an orphan receptor belonging to the TNF receptor superfamily. 5. Shao, Z. et al. (2005) Neuron 45:353. NgR1 is also bound by the myelin-derived inhibitors myelin-associated 6. Cao, Z. et al. (2010) Mol. Cell. Neurosci. 43:1. glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) 7. Atwal, J.K. et al. (2008) Science 322:869. (Figure 15C).6 MAG binds with higher affi nity to NgR2, but no ligand 8. Grunewald, E. et al. (2009) Mol. Cell. Neurosci. 42:363. has been identifi ed for NgR3. Further studies suggest that the Nogo-66 domain binds to Paired-Ig-like Receptor B (PIR-B).7 The binding partner 9. Neiderost, B. et al. (2002) J. Neurosci. 22:10368. for the Nogo-20 region remains to be confi rmed, but the G protein- 10. Joset, A. et al. (2010) J. Cell. Biol. 188:271.

A. B. C. Oligodendrocyte Nogo-A Oligodendrocyte MAG OMgp Nogo-'20 Nogo-66

N-term

Pincher-mediated endocytosis Growth Cone Signalosome

to the Rho-GTP cell body

NgR1 PIR-B NgR1 Neuron LINGO-1 NgR2 NGF R TROY GPR50 NGF R NgR3

Domain Key: Cysteine-rich Death domain Leucine-rich GPI anchor Ig-like ITIM Serine/Threonine-rich TNF R cysteine motif

Figure 15. Nogo Proteins and Receptors. A. Nogo-A expressed on oligodendrocytes binds to neuronal receptors via the Nogo-66 and Nogo-20 domains. B. Following binding to the tripartite complex of NgR1, NGF R, and LINGO-1 (or TROY), Nogo-A is endocytosed in a Pincher-dependent manner.10 Rho-GTP coated signalosomes are transported to the cell body where cyclic AMP response element-binding (CREB) phosphorylation is decreased and the transcription of growth genes is suppressed. Proteins containing multiple leucine-rich repeats are known to adopt a concave structure.5 C. MAG and OMgp also bind PIR-B (not shown).8 Coreceptors for NgR2 and NgR3, and the binding partner for NgR3 are yet to be determined.

24 For research use only. Not for use in diagnostic procedures. NEURAL DEVELOPMENT & AXON GUIDANCE

R&D Systems Products for Nogo Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

GPR50 H (FC, IHC) Nogo-B H (IHC, WB) M (IHC, WB)

LINGO-1 H (FC, IHC, WB) Nogo-C H (IHC, WB)

LINGO-2 H (FC, IHC, WB) OMgp H M H (WB) M (WB)

MAG/Siglec-4a RR (B/N, ELISA, IHC, WB) R PIR-B M (FC, WB)

NGF R/TNFRSF16 H M H (FC, IHC, WB) M (IHC, WB) ROCK1 HH (WB) M (WB) R (WB)

NgR2/NgRH1 H (WB) ROCK2 H (WB) M (WB) R (WB)

NgR3/NgRH2 H (WB) RTN1-A/NSP R (IHC)

Nogo Receptor/NgR H M H (B/N, WB) M (WB) TROY/TNFRSF19 H M H (WB) M (ELISA, IHC, WB) M

Nogo-A H R H (IHC, WB) R (IHC, WB) WNK1 H (WB) M (WB) R (WB) H M R

kDa N C2C 117

55 Nogo B 37

Nogo-A in Diﬀ erentiated Rat Cortical Stem Cells. Nogo-A was detected in Detection of Mouse Nogo-B by Western Blot. Western blot shows lysates of Nogo-C in Human Medulla. Nogo-C was detected in immersion-fi xed paraffi n- immersion-fi xed 7 day diff erentiated rat cortical stem cells using a Sheep Anti- the Neuro-2A mouse neuroblastoma cell line and the C2C12 mouse myoblast cell embedded sections of human brainstem using a Sheep Anti-Human Nogo-C Human Nogo-A Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF3515). line. The PVDF membrane was probed with a Sheep Anti-Mouse Nogo-B Antigen Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5766). The tissue was The cells were stained with the NorthernLights™ 557-conjugated Donkey Anti- Affi nity-purifi ed Polyclonal Antibody (Catalog # AF6596) followed by a Donkey HRP- stained using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS019; Sheep IgG Secondary Antibody (Catalog # NL010; red) and counterstained with DAPI conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specifi c band brown) and counterstained with hematoxylin (blue). Specifi c staining was localized (blue). was detected for Nogo-B at approximately 50 kDa (as indicated). to gigantocellular neurons in brainstem medulla.

Relative Cell Number Cell Relative 10

0 100 101 102 103 104 LINGO-1

MAG/Siglec-4a in Rat Cerebellum. Myelin-associated Glycoprotein (MAG)/ Detection of LINGO-1 by Flow Cytometry. The CHP-100 human neuroblastoma cell Nogo-B in Rat Embryonic Spinal Cord. Nogo-B was detected in immersion-fi xed Siglec-4a was detected in perfusion-fi xed frozen sections of rat brain using a Goat line was stained with a Mouse Anti-Human LINGO-1 Monoclonal Antibody (Catalog frozen sections of rat embryo (E13.5) using a Sheep Anti-Mouse Nogo-B Antigen

Anti-Rat MAG/Siglec-4a Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # # MAB30861, fi lled histogram) or a Mouse IgG1 Isotype Control (Catalog # MAB002, Affi nity-purifi ed Polyclonal Antibody (Catalog # AF6596). The tissue was stained AF538). The tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining open histogram), followed by an APC-conjugated Goat Anti-Mouse IgG Secondary using the NorthernLights 557-conjugated Donkey Anti-Sheep IgG Secondary Kit (Catalog # CTS008; brown) and counterstained with hematoxylin (blue). Specifi c Antibody (Catalog # F0101B). Antibody (Catalog # NL010; red) and counterstained with DAPI (blue). Specifi c labeling was localized to processes of oligodendrocytes. staining was localized to the white matter of developing spinal cord.

www.RnDSystems.com/go/Nogo 25 NEURAL DEVELOPMENT & AXON GUIDANCE

Bone Morphogenetic Proteins & Receptors Bone morphogenetic proteins (BMPs) represent a large subfamily of the cone that are independent of the nucleus. Receptor activation in growth Transforming Growth Factor- (TGF-) superfamily. Currently, there are cones induces an increase in intracellular calcium concentration via approximately twenty ligand members. These secreted signaling mole- Transient Receptor Potential Channel 1 (TRPC1).5 Elevated calcium levels cules exert important actions during embryogenesis and morphogenesis, activate Calcineurin, which stimulates the phosphatase Slingshot. In turn, including eff ects on cell proliferation, diff erentiation, and migration. In the Slingshot dephosphorylates and activates Actin depolymerizing proteins, adult, BMPs are known to aff ect bone and cartilage formation, vascular such as Cofi lin and ADF, triggering Actin depolymerization and growth homeostasis, and iron and glucose metabolism. Similar to the Hedgehog cone collapse (Figure 16).5 and Wnt morphogen families, studies indicate that BMPs also infl uence In addition, BMPs mediate attractive guidance cues through the activation axon guidance.1 of LIM Kinase 1 (LIMK1) and LIMK2. LIM kinases phosphorylate and Cleaved from precursors and secreted, BMP ligands function as homo- inactivate Cofi lin and ADF, facilitating Actin polymerization and growth or heterodimeric disulphide-linked proteins. Similar to other TGF- cone attraction.6,7 Thus, BMPs induce independent pathways to control superfamily ligands, BMPs bind to heterotetrameric receptor complexes Actin polymerization and exert opposing eff ects on axon guidance. Unlike composed of two type I and two type II receptors. Both types of receptor Hedgehog and Wnt family morphogens, BMPs do not require additional are single-pass transmembrane proteins with extracellular ligand-binding non-canonical receptor mechanisms to exert guidance versus cell fate and intracellular serine/threonine kinase domains. In general, BMP ligands actions. Instead, the molecular subunits that compose the canonical have a higher affi nity for type I receptors. Formation of a ligand-receptor receptor complex determine the diverse activities of BMPs. type I complex increases the affi nity for subsequent BMP binding to type- Although type I and type II receptors are thought to be equally important II receptors. Constitutively active type II receptors then phosphorylate for determining cell fate, BMPR-IB alone was both necessary and suffi cient type I receptors, which propagate signal transduction by activating Smad for the repellent guidance of chick dorsal commissural axons.8 Further- intracellular eff ector molecules. Interestingly, Repulsive Guidance Mole- more, eff ects on dorsal neuron cell fate specifi cation are governed by BMP cules (RGMs) have been shown to act as high-affi nity co-receptors for homodimers, whereas BMP7:GDF7 heterodimers control axon guidance. BMPs, but the physiological signifi cance of this interaction is unclear.2 Diff erential roles for BMPR-I subtypes is supported by a recent paper, which In the developing spinal cord, the ventrally orientated growth of dorsal reported BMPR-IA expression in the cell bodies and BMPR-IB expression commissural axons is initiated by BMP-7 and GDF-7 chemorepellents in the dendrites of adult rat neurons.9 In Drosophila, the BMP homolog derived from the roof plate.3,4 Although BMPs modulate cell fate via Glass bottom boat (Gbb) has been shown to induce synaptogenesis at global changes in transcription, eff ects on axon guidance are dependent neuromuscular junctions through retrograde signaling, but this action is on local activation of signal transduction pathways in the axonal growth yet to be reported in mammalian neuronal systems.10,11 References

1. Sánchez-Camacho, C. & P. Bovolenta (2009) BioEssays 31:1013.

2. Corradini, E. et al. (2009) Cytokine Growth Factor Rev. 20:389. BMP-7:GDF-7 3. Butler, S.J. & J. Dodd (2003) Neuron 38:389. Ca2+ Extracellular 4. Augsburger, A. et al. (1999) Neuron 24:127. BMPR-II BMPR-I TRPC1 5. Wen, Z. et al. (2007) J. Cell. Biol. :107. Plasma Membrane 178 6. Foletta, V.C. et al. (2003) J. Cell. Biol. 162:1089. Intracellular 7. Ng, J. (2008) Development 135:4025. Ca2+ m 8. Yamauchi, K. et al. (2008) Development 135:1119. LIMK1/2 Calcineurin 9. Miyagi, M. et al. (2011) Neuroscience 176:93.

10. Ball, R.W. et al. (2010) Neuron 66:536. LIMK1/2 Slingshot 11. Nahm, M. et al. (2010) J. Neurosci. 30:8138.

Inactivation ADF Cofilin

ADF Cofilin ADF Cofilin

Actin Polymerization Actin Depolymerization This specialized catalog features a restricted list of products & Neurite Outgrowth & Growth Cone Collapse ATTRACTION REPULSION for BMP-related molecules. For a complete list of related products, please visit: www.RnDSystems.com/go/BMPs Figure 16. Transcription-independent Axon Guidance by BMPs. Heterodimers of BMP-7:GDF-7 exert bidirectional guidance cues via modulation of the Actin cytoskeleton. Activation of LIMK1/2 inactivates ADF and Cofi lin, promoting Actin polymerization and neurite outgrowth. In contrast, interactions between BMPR-II and TRPC1 increase intracellular calcium concentration and activate Slingshot, which dephosphorylates LIMK1/2 leading to disassembly of the cytoskeleton and growth cone collapse.

26 For research use only. Not for use in diagnostic procedures. NEURAL DEVELOPMENT & AXON GUIDANCE

R&D Systems Products for BMP Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

BMPs (Bone Morphogenetic Proteins) Cerberus 1 MH (IHC, WB) M (WB)

BMP-2 H Z H (B/N, ELISA, IHC, WB) Z (B/N, WB) H M R Chordin MM (B/N, ELISA, IHC, WB) M

BMP-2/BMP-7 HH (WB) Chordin-like 1/ HH (WB) Heterodimer CHRDL1

BMP-2/BMP-4 H (B/N, IHC, WB) Z (WB) Chordin-like 2/ MH (FC, IHC, IP, WB) M (IHC, IP, WB) CHRDL2 BMP-2a Z COCO H M H (ELISA, WB) M (WB) H BMP-3 HH (IHC, WB) Crossveinless-2/CV-2 H M H (IHC, WB) M (IHC, WB) BMP-3b/GDF-10 HH (IHC, WB) DAN H M H (B/N, ELISA, IHC, WB) M (B/N, ELISA, WB) H M BMP-4 H M Z H (B/N, ELISA, IHC, WB) Z (WB) H Follistatin H M H (B/N, IHC, WB) M (WB) H BMP-4/BMP-7 H Heterodimer Follistatin-related H M H (B/N, ELISA, WB) M (B/N, IHC, WB) H Gene Protein/FLRG BMP-5 H M H (B/N, ELISA, IHC, WB) M (IHC, WB) H Gremlin H M M (B/N, ELISA, IHC, WB) M BMP-6 H M H (B/N, ELISA, IHC, WB) M (IHC, WB) H Noggin H M M (IHC, WB) BMP-7 H M H (B/N, ELISA, IHC, WB) H PRDC/GREM2 MM (ELISA, WB) M BMP-8 H (IHC, WB) SOST/Sclerostin H M H (ELISA, WB) M (IHC, WB) H BMP-8a H TMEFF1/ H (WB) M (IHC, WB) BMP-9 H M H (B/N, ELISA, WB) M (B/N, ELISA, WB) H Tomoregulin-1

BMP-10 H M H (B/N, WB) M (B/N, IHC, WB) TSG MM (IHC, WB)

BMP-15/GDF-9B HH (IHC, WB) M (IHC, WB) R (WB) Tsukushi/TSK HH (IHC) M (WB)

BMP-8b H (WB) M (WB) USAG1 HH (WB) Decapentaplegic/ DD (WB) Growth/Diﬀ erentiation Factors (GDFs) DPP GDF-1 HM (WB) BMP Receptors GDF-3 H M H (IHC, WB) M (IHC, WB) BMPR-IA/ALK-3 H M H (FC, IHC, WB) GDF-5/BMP-14 MM (B/N, IHC, WB) BMPR-IB/ALK-6 H M H (FC, IHC, WB) M (WB) GDF-6/BMP-13 M BMPR-II H M H (FC, IHC, WB) GDF-7/BMP-12 MM (WB) BMP Modulators GDF-8/Myostatin H M R H (B/N, IHC, WB) M (B/N, IHC, WB) R (B/N, IHC, WB) BAMBI/NMA H (IHC, WB) M (WB) GDF-9 MM (IHC, WB) BMP-1/PCP HH (IHC, IP, WB) GDF-11/BMP-11 H Brorin/VWC2 HH (IHC, WB) M (IHC, WB) GDF-15 HH (ELISA, WB) M (IHC) H Caronte Ch Ch (B/N, WB)

BMP-7 Antibody (Pg/mL) kDa Mo Br 10-1 100 101 102 2.0 2.0 94 65 Cell Proliferation (Mean O.D.) 39 1.5 1.5

1.0 1.0 23 19 Calcineurin B 0.5 0.5 Cell Proliferation (Mean O.D.) Proliferation Cell

0.0 0.0 6 10-2 10-1 100 101 BMP-7 (Pg/mL)

Alkaline Phosphatase Production Induced by BMP-7 and Neutralization by Detection of Mouse Calcineurin B by Western blot. Western blot shows lysates of BMPR-II in Human Prostate. BMP Receptor II (BMPR-II) was detected in a BMP-7 Antibody. Recombinant Human BMP-7 (Catalog # 354-BP) induces mouse brain tissue. The PVDF membrane was probed using a Rabbit Anti-Human/ immersion-fi xed paraffi n-embedded sections of human prostate using a Goat Anti- alkaline phosphatase production in the ATDC5 mouse chondrogenic cell line in a Mouse/Rat Calcineurin B Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # Human BMPR-II Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF811). dose-dependent manner (green line). Alkaline phosphatase production elicited AF1348) followed by a HRP-conjugated Goat Anti-Rabbit IgG Secondary Antibody The tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit by Recombinant Human BMP-7 (1 g/mL) is neutralized (gray line) by increasing (Catalog # HAF008). A specifi c band was detected for Calcineurin B at approximately (Catalog # CTS008; brown) and counterstained with hematoxylin (blue). Specifi c concentrations of a Mouse Anti-Human BMP-7 Monoclonal Antibody (Catalog # 17 kDa (as indicated). labeling was localized to the plasma membrane of epithelial cells. MAB3541). www.RnDSystems.com/go/BMPs 27 NEURAL DEVELOPMENT & AXON GUIDANCE

Repulsive Guidance Molecules & Receptors In mammals, the Repulsive Guidance Molecule (RGM) family consists of RGM-B, also know as DRAGON (turned ON in DRG), is expressed throughout three glycoproteins that have discrete expression patterns and functions the CNS in an almost non-overlapping distribution to RGM-A. Although (RGM-A, RGM-B, and RGM-C).1 A fourth member, RGM-D, is only expressed RGM-B is a homolog of RGM-A, it has been shown to exert no repulsive in fi sh. RGMs share common structural domains, including a N-terminal action on migrating axons.10 Instead, recent studies reported that RGM-B signal sequence, a partial von Willebrand factor (vWF) type D domain, and inhibits neuronal migration in the developing dentate gyrus and that this a C-terminal glycosylphosphatidylinositol (GPI) anchor. RGM proteins can action is dependent on Neogenin.11 RGM-C/Hemojuvelin is not expressed exist as single chain proteins or two chains linked by disulphide bridges, in the CNS. Mutations in the gene that encodes RGM-C result in juvenile following cleavage at a highly conserved site within the vWF domain. In hemochromatosis, an autosomal-recessive disease characterized by early addition, RGM-A and RGM-C also contain a RGD (Arg-Gly-Asp) motif, an onset systemic iron overload.12 RGM-C is thought to control the expression Integrin binding site thought to facilitate cell adhesion. of hepcidin, a key regulator of iron metabolism in hepatocytes. RGMs are known to function through Neogenin, a homologue of the Netrin References receptor Deleted in Colon Cancer (DCC). Like DCC, Neogenin is a type I 1. Severyn, J. et al. (2009) Biochem. J. 422:393. transmembrane protein with four immunoglobulin and six fi bronectin type 2. De Vries, M. & H.M. Cooper (2008) J. Neurochem. 106:1483. III repeats in the extracellular sequence, and three conserved intracellular 3. Rajagopalan, S. et al. (2004) Nat. Cell. Biol. 6:755. domains (P1-3). Neogenin is a multifunctional receptor that can mediate Netrin-induced attraction and RGM-induced repulsion (Figure 18).2 RGMs 4. Monnier, P.P. et al. (2002) Nature 419:392. were shown to bind the fi fth and sixth fi bronectin repeats, whereas Netrin 5. Hata, K. et al. (2009) J. Cell. Biol. 184:737. 3 is believed to interact with the fourth and fi fth fi bronectin repeats. It is not 6. Hata, K. et al. (2006) J. Cell. Biol. 173:47. clear whether Netrin and RGM binding to Neogenin is mutually exclusive, 7. Niederkofl er, V.R. et al. (2004) J. Neurosci. 24:808. but because Neogenin has a greater binding affi nity for RGM, repulsion would override attraction if a growth cone simultaneously encountered 8. Koeberle, P.D. et al. (2010) Neuroscience 169:495. both guidance cues. 9. Bradford, D. et al. (2010) J. Comp. Neurol. 173:47. RGM-A was discovered in 2002 as a chemorepulsive signal for temporal 10. Samad, T.A. et al. (2010) J. Neurosci. 24:2027. retinal axons in chick anterior tectum.4 In this system, an anterior-high, 11. Conrad, S. et al. (2010) Mol. Cell. Neurosci. 43:222. posterior-low gradient of RGM-A prevents Neogenin-positive neurons 12. Corradini, E. et al. (2009) Cytokine Growth Factor Rev. 20:389. from entering the anterior tectum and drives projections into the posterior territory. In addition to Neogenin, recent studies suggest that RGM-A induced growth cone collapse requires coreceptor UNC5, and that signal Intracellular 5 transduction involves the Leukemia-associated RhoGEF (LARG) (Figure 17). Plasma Membrane Additional studies have shown that RGM-A expression inhibits axon RGM-A regeneration following injury, an eff ect that can be neutralized by Neogenin RGM-A antibody administration.6 RGM-A is also known to aff ect neural Neogenin tube closure, with loss of function mice exhibiting exencephaly, a severe developmental defect.7 Other studies suggested that Neogenin is a Extracellular dependence receptor, and that RGM-A promotes cell survival by inhibiting Netrin-1 UNC5 Neogenin-induced apoptotic cell death.8 Because RGM-A and Neogenin are expressed in neurogenic areas of mouse and human adult forebrain, RGM-A signaling has also been implicated in neurogenesis.9 Plasma Membrane

1 1 Intracellular 2 2 LARG Src 3 SHP 3 A. B. FAK FAK Rac-1 RhoA ROCK

NEURITE OUTGROWTH GROWTH CONE COLLAPSE & ATTRACTION & REPULSION

Domain Key: Laminin-like EGF repeat Heparin-binding Ig-like Conserved IC GPI anchor vWF RGD

Fibronectin type III Thrombospondin-like ZU5 DCC binding Death domain

Figure 17. RGM-A-induced Growth Cone Collapse. A. An embryonic rat cortical growth cone (E19-20) cultured under control Figure 18. Neogenin Mediates Attractive and Repulsive Guidance Cues. Netrin-1 binds Neogenin to promote neurite outgrowth conditions. B. Treatment with recombinant mouse RGM-A (Catalog # 2458-RG; 2 μg/mL) induces growth cone collapse. Figure and chemoattraction via Rac-1. RGM-A interacts with Neogenin which, through the UNC5 co-receptor and FAK, phosphorylates adapted with permission from Hata, K. et al. (2009) J. Cell Biol. 184:737. LARG. Subsequent activation of the RhoA/ROCK pathway results in growth cone collapse and axon repulsion.5 Netrin-1 can also induce axon repulsion through UNC5 (please see page 14).

28 For research use only. Not for use in diagnostic procedures. NEURAL DEVELOPMENT & AXON GUIDANCE

R&D Systems Products for RGM Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

DAPK3/ZIPK H (WB) ROCK1 HH (WB) M (WB) R (WB)

DCC MH (IHC, WB) M (B/N, WB) ROCK2 H (WB) M (WB) R (WB)

FAK HH (IHC, WB) M (IHC, WB) R (IHC, WB) H M R SHP-1 HH (IHC, WB) M (IHC, WB) R (IHC, WB) H M R

LARG H (WB) SHP-2 HH (IHC, WB) M (IHC, WB) R (WB) H M R

Neogenin MM (B/N, IHC, WB) Src H V H (IHC, WB) M (IHC, WB) R (IHC, WB) H

Netrin-1 H M Ch M (B/N, WB) Ch (WB) UNC5H1 H R R (B/N, IHC, WB)

RGM-A H M H (IHC, WB) M (IHC, WB) Ch (WB) M R UNC5H2 RR (IHC, WB)

RGM-B H M H (FC, WB) M (FC, IHC, WB) UNC5H3 HH (IHC, WB)

RGM-C/Hemojuvelin H M H (WB) M (WB) UNC5H4 HH (WB)

A. - B. at

kDa NR-

kDa R 209 122 120 94 99 65 Src SHP-2 53 39

38 23 19 19

7 7

RGM-B in Mouse Trigeminal Ganglia. Repulsive Guidance Molecule-B (RGM-B) Detection of Rat Src and Mouse SHP-2 by Western Blot. Western blot shows FAK in Human Hippocampus. Focal Adhesion Kinase 1 (FAK) was detected in was detected in perfusion-fi xed frozen sections of mouse brain using a Sheep Anti- lysates of the Rat-2 rat embryonic fi broblast cell line and the NR-6 mouse fi broblast immersion-fi xed paraffi n-embedded sections of human hippocampus using a Mouse RGM-B Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF3597). The cell line. A. The PVDF membrane was probed with a Goat/Anti-Human/Mouse/Rat Sheep Anti-Human/Mouse/Rat FAK Antigen Affi nity-purifi ed Polyclonal Antibody tissue was stained with the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (Catalog Src Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF3389) followed by (Catalog # AF4467). The tissue was stained with the Anti-Sheep HRP-DAB Cell & # CTS019; brown) and counterstained with hematoxylin (blue). a HRP-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # HAF109). Tissue Staining Kit (Catalog # CTS019; brown) and counterstained with hematoxylin B. The PVDF membrane was probed with a Goat Anti-Human/Mouse/Rat SHP-2 (blue). Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF1894) followed by a HRP- conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # HAF109). Src and SHP-2 were detected at approximately 60 kDa and 67 kDa, respectively (as indicated). Additional Axon Guidance Molecules R&D Systems off ers additional products for molecules that are known to eff ect specifi c aspects of axon guidance but are not traditionally considered to be members of the protein families presented in this catalog.

R&D Systems Products for Additional Axon Guidance Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Cadherin-10 H MDGA1 HH (WB)

Draxin H M H (IHC, WB) M (IHC, WB) R (IHC, WB) MDGA2 HH (WB)

EMP3 H (FC) Mena H (WB)

F-Spondin/SPON1 HH (B/N, WB) Neurocan H M M (IHC, WB) R (IHC, WB)

GAP-43 H (WB) M (WB) R (WB) B (WB) Ca (WB) Ch (WB) Neurofascin RH (IHC, WB) M (IHC, WB) R (IHC, WB) Pr (WB) X (WB) Z (WB) Ninjurin-1 H (WB) Katanin p60 H (IHC) Ninjurin-2 H (IHC, WB) Kilon/NEGR1 HH (WB) M (WB) PRG-1/LPPR4 H (WB) LRRN1/NLRR-1 H (IHC, WB) M (IHC, WB) Stathmin-2/STMN2 H (IHC, WB) M (IHC, WB) LRRN3/NLRR-3 H

www.RnDSystems.com/go/RGM 29 CELL ADHESION MOLECULES FOR NEUROSCIENCE

Contactin Family Contactins compose a subgroup of the Immunoglobulin (Ig) superfamily Contactin-2 is expressed on subsets of neurons in the spinal cord, dorsal of cell adhesion molecules (IgCAM). This molecule group consists of root ganglion, and retinal ganglion cells. Expression of Contactin-2 Contactin-1, Contactin-2/TAG-1, Contactin-3/BIG-1, Contactin-4/BIG-2, is essential for the tangential migration of GABA-positive cortical Contactin-5/NB2, and Contactin-6/NB3.1 Members of the Contactin family interneurons from the medial ganglionic eminence of the ventral share a common molecular structure, which includes six Ig-like domains, telencephalon.5 Contactin-2 also mediates the superfi cial migration of four fi bronectin type-III domains, and a glycosylphosphatidylinositol neuronal populations that form several precerebellar nuclei through (GPI) anchor. Contactins exert critical eff ects during development and the superfi cial and olivary migratory streams to the caudal medulla.6 In in the adult nervous system. Their actions infl uence essential biological addition, knockout of Contactin-2 results in aberrant chemorepulsion of processes including neural migration, adhesion, and diff erentiation, and somatosensory aff erent neurons in the dorsal horn.7 neurite outgrowth and fasciculation. Contactin-1 and Contactin-2 are known to play important roles at With the exception of Contactin-2, which is expressed during the specialized domains located in myelinated axons. At nodes of development, Contactin expression commences postnatally. Because Ranvier, one micrometer long interruptions in the myelin sheath, Contactins are expressed on axons and are known to promote neurite Contactin-1 binds to the 1 subunit of voltage-gated sodium channels outgrowth, initial studies investigated the role of Contactins in axon to enhance their cell surface expression.8 On the axon plasma membrane guidance and cell migration. In the postnatal cerebellum, Contactin-1 (axolemma), Contactin-1 binds Contactin-associated Protein (Caspr) and is expressed on migratory granule cells. Interestingly, the subcellular Neurofascin-155 to promote the formation of paranodes, myelinated expression of Contactin-1 changes during migration. Expression levels regions that fl ank nodes of Ranvier.9 Furthermore, glial Contactin-2 are decreased in the cell body but maintained on axonal extensions in the interacts with axonal Caspr2 and voltage-gated potassium channels to molecular layer.2 Genetic knockout experiments suggest that Contactin-1 maintain the juxtaparanode domain and ensure rapid action potential controls axodendritic interactions between cerebellar interneurons, propogation.10 and is required for normal cerebellar morphogenesis.3 Recent studies Less is known about the physiological roles of Contactins-3 through -6. suggest that the sequential expression of Contactin-2 and Contactin-1 In adult brain, Contactin-3 is expressed in specifi c neuron populations, regulates Sonic Hedgehog (Shh)-induced proliferation and diff erentiation including Purkinje cells in the cerebellum, granule cells in the dentate 4 of cerebellar granule neuron progenitor cells. This research found that gyrus of the hippocampus, and neurons in the superfi cial layer of the Contactin-1 inhibits Shh dependent proliferation and promotes neuronal cerebral cortex.11 Contactin-4 is believed to orchestrate the convergence diff erentiation via the L1-like NgCAM-related cell adhesion molecule of olfactory sensory neurons and is crucial for the formation and (NrCAM) (Figure 19). In contrast, Contactin-2 binds NrCAM to antagonize maintenance of the odor map in the olfactory bulb.12 Highly restricted to Contactin-1 and facilitate cell proliferation. specifi c brain regions, Contactin-5 knockout impairs auditory brainstem function by decreasing synapse formation and inducing neuronal Intermediate Granule oEGL apoptosis.13 Similarly, genetic deletion of Contactin-6 specifi cally reduced Neuron Progenitors mEGL the formation of glutaminergic synapses in the cerebellum, supporting Sonic Hedgehog iEGL the hypothesis that Contactin-6 modulates cerebellar control of motor coordination.14 A recent study showed that Contactin-1 binds Protein ML Tyrosine Phosphatase Receptor  (PTPR) and Contactins-3 through -6 bind to PTPR, presenting a novel mechanism by which Contactins may IGL induce intracellular signaling.15

NrCAM Contactin-2Contactin-1 NrCAM Contactin-1 References 1. Shimoda, Y. & K. Watanabe (2009) Cell Adhesion & Migration 3:64.

2. Virgintino, D. et al. (1999) J. Comp. Neurol. 413:357.

3. Berglund, E.O. et al. (1999) Neuron 24:739.

4. Denaxa, M. et al. (2001) Development 128:4635.

5. Kyriakopoulou, K. et al. (2002) Development 197:287.

6. Law, C.O. et al. (2008) Development 135:2361.

7. Xenaki, D. et al. (2008) Development 138:519.

CELL CYCLE ENTRY CELL CYCLE EXIT 8. Swanwick, R.S. et al. (2010) Neurosci. Lett. 486:78. & PROLIFERATION & DIFFERENTIATION 9. Pomicter, A.D. et al. (2010) Brain 133:389.

10. Sawaki, M. et al. (2010) J. Neurosci. 30:13943.

11. Yoshihara, Y. et al. (1994) Neuron 13:415. Domain Key: Ig-like Fibronectin type III GPI anchor 12. Kaneko-Goto, T. et al. (2008) Neuron 57:834.

Figure 19. Antagonistic Eﬀ ects of Contactins on Cerebellar Granule Neuron Precursors. In the medial external germinal layer 13. Toyoshima, M. et al. (2009) Dev. Biol. 336:192. (mEGL) of the developing cerebellum, intermediate granule neuron progenitors proliferate in response to Purkinje cell-derived Sonic Hedgehog. Contactin-2 promotes proliferation by preventing interactions between Contactin-1 and NrCAM. Following 14. Sakurai, K. et al. (2010) Neurosci. Lett. 473:102. migration to the inner EGL (iEGL), and in the absence of Contactin-2, Contactin-1 binds to NrCAM to stimulate cell cycle exit and 15. Bouyain, S. & D.J. Watkins (2010) Proc. Natl. Acad. Sci. USA 107:2443. neuronal diff erentiation. Abbreviations: outer external germinal layer (oEGL), inner granule layer (IGL), molecular layer (ML).

30 For research use only. Not for use in diagnostic procedures. CELL ADHESION MOLECULES FOR NEUROSCIENCE

R&D Systems Products for Contactin Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs Contactins Related Molecules

Contactin-1 HH (B/N, IHC, WB) Caspr2 H (IHC, WB)

Contactin-2/TAG1 H M H (IHC, WB) M (IHC, WB) R (IHC, WB) Neurofascin RH (IHC, WB) M (IHC, WB) R (IHC, WB) H

Contactin-3 HH (FC, WB) M (WB) R (WB) NrCAM HH (ELISA, IHC, WB)

Contactin-4 H M H (B/N, WB) M (IHC, WB) PTPR  H (IHC, WB), M (WB), R (WB)

Contactin-5 HH (WB) PTPRZ H (IHC, WB)

Contactin-6 MM (IHC, WB)

e x s te u or C kDa Mo A. B. 204 Contactin-3 117

Retinal Ganglion Cell (RGC) Axon Fasciculation Requires Homophilic Detection of Mouse Contactin-3 by Western Blot. Western blot shows lysates of Contactin-2/TAG1 in Human Cortex. Contactin-2/TAG1 was detected in immersion Contactin-2/TAG1 Interactions. Retinal explants were isolated from E14.5 mouse cortex. The PVDF Membrane was probed with a Rat Anti-Mouse Contactin-3 fi xed paraffi n-embedded sections of human cortex using a Mouse Anti-Human TAG1+/– or TAG1–/– embryos. In vitro assays of RGC axonal growth were Monoclonal Antibody (Catalog # MAB6644) followed by a HRP-conjugated Goat Contactin-2/TAG1 Monoclonal Antibody (Catalog # MAB1714). The tissue was performed on glass coverslips coated with alternating stripes of Laminin or Anti-Rat IgG Secondary Antibody (Catalog # HAF005). Contactin-3 was detected at stained using the Anti-Mouse HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS002; Recombinant Human Contactin-2/TAG1 (Catalog # 1714-CN). A. TAG1+/– axons approximately 125 kDa (as indicated). brown) and nuclei were counterstained with hematoxylin (blue). Specifi c staining prefer Contactin-2/TAG1 to Laminin. B. Contactin-2/TAG1–/– axons display no was localized to the cytoplasm, plasma membrane, and processes of cortical preference between Contactin-2/TAG1- and Laminin-coated stripes. RGC axons were neurons. immunolabeled with anti-neurofi lament antibodies (red).Data Courtesy of Dr. Jean- Léon Thomas, Université Pierre et Marie Curie, Paris.

www.RnDSystems.com/go/Contactins 31 CELL ADHESION MOLECULES FOR NEUROSCIENCE

Nectins & Cadherins Ubiquitously expressed throughout the CNS, Nectins are immunoglobulin pyramidal dendrites require presynaptic Nectin-1 and postsynaptic (Ig)-like cell adhesion molecules that exert multiple eff ects on cell migration, Nectin-3.5 Some neuronal actions of Nectin molecules are known to be proliferation, survival, and diff erentiation. Members of the Nectin family independent of Cadherin recruitment. For example, guidance of com- (Nectin-1 to -4) are structurally characterized by three extracellular Ig-like missural axons across the midline of the spinal cord is dependent on domains, a single transmembrane region, and an intracellular C-terminal the expression of Nectin-3 on the migrating axons and Nectin-1 on the PDZ domain. Nectins preferentially form heterophilic complexes to dendrites of fl oor plate cells.6 In addition, Nectin-3 is important for the promote calcium-independent cell-cell adhesion through trans inter- correct targeting of callosal projection neurons, cortical commissural action of their extracellular domains. To facilitate synaptic adhesion, axons that connect homotropic brain regions via the corpus callosum.7 Nectin molecules are anchored to the Actin cytoskeleton via a PDZ- Recent studies suggest that activity-dependent cleavage of Nectin-1 dependent interaction with Afadin, which in turn binds F-Actin. During induces dendritic spine remodeling and may represent a novel mode of the generation of new synapses, Nectins are believed to form a relatively synaptic plasticity.8 Kim et al. discovered that NMDA receptor activation weak adhesion complex, which is strengthened following the recruitment caused proteolytic processing of Nectin-1 by ADAM10 and -Secretase, an of Cadherin molecules.1 eff ect that was dependent on calcium infl ux and Calmodulin (Figure 20).8 Cadherins are single-pass transmembrane cell adhesion molecules with a ADAM10 also aff ects dendritic spine morphology through cleavage of variable number of extracellular calcium-binding cadherin repeats. Unlike Neuronal Cadherin (N-Cadherin), which is highly expressed in neural Nectin molecules, Cadherins predominantly associate as homophilic tissues.9 In primary cultures of hippocampal neurons, N-Cadherin- complexes and mediate cell-cell adhesion in a calcium-dependent Catenin complexes colocalize with Nectin-Afadin prior to the formation of manner.2 Furthermore, the binding of calcium ions to the cadherin excitatory synapses, supporting the theory that both adhesion systems are repeats enhances dimerization and galvanizes intercellular connections required for synaptogenesis.10 Recent studies also suggest that N-Cadherin by increasing resistance to proteolytic degradation.3 In parallel to the promotes glutamatergic synapse formation by recruiting members of the Nectin-Afadin adhesion system, the intracellular Cadherin region interacts Neuroligin adhesion molecule family.11 In addition, bidirectional coupling with -Catenin, which fastens to the Actin cytoskeleton via -Catenin. The of N-Cadherin and Neuroligin-1 signaling was shown to induce presynaptic trans interaction of Nectin molecules across the synapse is believed to vesicle clustering at nascent synapses.12 Additional studies showed that stimulate the recruitment of Cadherin molecules following the activation N-Cadherin-dependent reorganization of the Actin cytoskeleton was of a c-Src non-receptor tyrosine kinase.4 Src activation modulates Actin essential for metabotropic Glutamate Receptor 2 (mGluR2)-dependent dynamics through downstream eff ectors (i.e. Rap1, IQGAP1, N-WASP, long-term depression (LTD).13 Furthermore, experiments using zebrafi sh and WASP), facilitating the binding of -Catenin and the recruitment of embryos support important developmental actions for N-Cadherin. Cadherin--Catenin complexes. Mutational studies revealed that N-Cadherin is critical for the guidance of In neurons, Nectin function has been most thoroughly characterized motor axons at the horizontal myoseptum and the control of progenitor 14,15 at puncta adherentia junctions (PAJ), mechanical adhesion sites that cell proliferation in the neural tube. fl ank the synaptic active zone (Figure 20). For example, in the adult hip- References pocampus, PAJ connections between mossy fi ber terminals and CA3 1. Ogita, H. et al. (2010) Proc. Jpn. Acad. 86:621.

2. Giagtzoglou, N. et al. (2009) Cold Spring Harb. Perspect. Biol. 1:a003079.

3. Shapiro, L. et al. (2007) Annu. Rev. Neurosci. 30:451. Axon Terminal SYNAPTIC 4. Fukuhara, T. et al. (2004) J. Cell. Biol. 166:393. VESICLE CLUSTERING 5. Mizoguchi, A. et al. (2002) J. Cell. Biol. 156:555. 6. Okabe, N. et al. (2004) Dev. Biol. 273:244.

7. Molyneaux, B.J. et al. (2009) J. Neurosci. 29:12343. Active Zone NMDA R 8. Kim, J. et al. (2010) J. Biol. Chem. 285:22919.

Dendritic 9. Malinverno, M. et al. (2010) J. Neurosci. 30:16343. Spine Integrin DvE3 2+ Neurexin uCa N-Cadherin N-Cadherin D-Secretase 10. Lim, S.T. et al. (2008) J. Comp. Neurol. 507:1228. ADAM10 11. Aiga, M. et al. (2011) J. Biol. Chem. 286:851. Nectin-3 Calmodulin Nectin-1 Neuroligin-1 12. Stan, A. et al. (2010) Proc. Natl. Acad. Sci. USA 107:11116. Nectin-1 13. Zhou, Z. et al. (2011) J. Neurosci. 31:819. Nectin-3 -Catenin S-SCAM D-Catenin D Puncta Adherentia Afadin E-Catenin 14. Brusés, J.L. et al. (2011) J. Comp. Neurol. 519:1797. E-Catenin Afadin 15. Chalasani, K. & R.M. Brewster (2011) Mol. Biol. Cell 22:1505. F-Actin Dendrite

Figure 20. Nectins and Cadherins Provide Axodendritic Adhesion. The Nectin-Afadin and Cadherin-Catenin adhesion systems are essential for the formation and maintenance of trans-synaptic connections. Recent studies report that Nectin-1 and N-Cadherin are cleaved by ADAM10.8,9 In addition, N-Cadherin recruits Neuroligin-1 to promote presynaptic vesicle clustering.11,12

32 For research use only. Not for use in diagnostic procedures. CELL ADHESION MOLECULES FOR NEUROSCIENCE

R&D Systems Products for Nectin & Cadherin Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs Nectins Desmocollin-1 H

Nectin-1 HH (FC, WB) Desmocollin-2 HH (IP, WB)

Nectin-2/CD112 H M H (FC, WB) M (WB) Desmocollin-3 HH (WB)

Nectin-3 HH (FC, IHC, WB) M (WB) Desmoglein-1 HH (IHC, WB)

Nectin-4 H M H (FC, IHC, WB) M (WB) Desmoglein-2 HH (WB)

Cadherin Superfamily Desmoglein-3 HH (IHC, WB)

Cadherin-4/R- H M H (WB) M (WB) Desmoglein-4 H (WB) Cadherin MUCDHL H (IHC, WB) M (FC, WB) Cadherin-6/KCAD H M H (FC, IHC, WB) Protocadherin-1 H (FC, IHC, WB) Cadherin-7 H Protocadherin-8 H (IHC, WB) Cadherin-8 HH (B/N, FC, WB) Protocadherin-10 H Cadherin-10 H Protocadherin α1 H (IHC, WB) Cadherin-11 H M H (B/N, FC, IHC, WB) Protocadherin α4 H Cadherin-12 HH (FC, IHC, WB) Protocadherin-15 H (IHC) Cadherin-13 H M H (FC, IHC, WB) Related Molecules Cadherin-17 H (IHC, WB) ADAMTS10 H (IP, WB) Cadherin-20 H β-Catenin H (ChIP, FC, IHC, WB) M (ChIP, FC, IHC, WB) H E-Cadherin H M H (ELISA, FC, IHC, WB) M (ELISA, FC, IHC, WB) H M R (ChIP, FC, IHC, WB) X (WB)

M-Cadherin/ HH (FC, IHC, WB) IQGAP1 H (WB) Cadherin-15 mGluR2 H (FC, WB) M (WB) R (WB) Ch (WB) Z (WB) N-Cadherin H M H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) N-WASP H (IHC, WB) P-Cadherin H M H (ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M Rap1A/B H (IHC, WB) M (IHC, WB) R (IHC, WB) VE-Cadherin H M H (FC, IHC, WB) M (FC, WB) H WASP H (IHC, WB) M (IHC, WB) CELSR2 H (IHC, WB)

kDa H C6 NIH-

100 E-Catenin 80 60 50 40

N-Cadherin in Human Endothelial Cells. N-Cadherin was detected in immersion- Detection of Human/Mouse/Rat -Catenin by Western Blot. Western blot shows N-Cadherin in Human Cortex. N-Cadherin was detected in immersion-fi xed fi xed paraffi n-embedded sections of human hippocampus using a Sheep Anti- lysates of the Huh-7 human hepatoma cell line, the C6 rat glioma cell line, and the paraffi n-embedded sections of human cortex using a Sheep Anti-Human/Mouse/ Human N-Cadherin Propeptide Antigen Affi nity-purifi ed Polyclonal Antibody NIH-3T3 mouse embryonic fi broblast cell line. The PVDF membrane was probed Rat N-Cadherin Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF6426). (Catalog # AF1388). The tissue was stained with the Anti-Sheep HRP-DAB Cell & with a Mouse Anti-Human/Mouse/Rat -Catenin Monoclonal Antibody (Catalog # The tissue was stained using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit Tissue Staining Kit (Catalog # CTS019; brown) and counterstained with hematoxylin MAB1329) followed by a HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody (Catalog # CTS019; brown) and counterstained with hematoxylin (blue). Specifi c (blue). Specifi c labeling was localized to endothelial cells in a capillary. (Catalog # HAF007). A specifi c band was detected for -Catenin at approximately staining was localized to neuronal cell bodies and processes. 95 kDa (as indicated).

www.RnDSystems.com/go/CellAdhesion 33 CELL ADHESION MOLECULES FOR NEUROSCIENCE

SynCAM, NCAM, & L1CAM Families The immunoglobulin (Ig) superfamily of cell adhesion molecules (IgCAM) Neural cell adhesion molecule (NCAM) was one of the fi rst proteins shown is an evolutionarily ancient and diverse group of proteins, several members to be involved in interneuronal adhesion. In neurons, one NCAM gene of which exert important neural actions via homotropic and heterotropic generates three isoforms, two single-pass transmembrane proteins and interactions. SynCAMs were fi rst identifi ed as calcium-independent a glycosylphosphatidylinositol (GPI)-linked protein. In cultured hippo- synapse-inducing molecules, but are now known to mediate myelination campal neurons, synaptogenesis required postsynaptic NCAM modifi ed by facilitating the interaction between Schwann cells and axons.1,2 Also with polysialic acid (PSA), and activation of NMDA and FGF receptors.8 referred to as Nectin-like molecules (Necl) 1-4 and Ig superfamily (IgSF) Recent studies suggest the activation of intracellular signaling molecules 4A-D, the diff erential expression pattern of SynCAM-1 to -4 is thought to by NCAM is dependent on its dimerization in cis (Figure 21)9. Other groups provide adhesion signals for neuronal circuit formation.3 have studied the eff ects of NCAM expression on neurite outgrowth. Cassens described a functional relationship between NCAM and TrkB, Although generally considered to infl uence the later stages of neural et al. in which BDNF stimulation of TrkB lead to NCAM phosphorylation and the circuit formation, recent studies report that SynCAMs also aff ect axon extension of hippocampal neurites.10 In parallel, proteolytic processing guidance. Niederkofl er et al. investigated the importance of SynCAM expression for the guidance of commissural axons in chick spinal cord. of NCAM and FAK was shown to be required for cerebellar neurite Their fi ndings indicate that the expression of SynCAM-2 on fl oor plate outgrowth. This eff ect was dependent on endosomal translocation of 11 cells is required for the rostral turning of axons following their crossing the intracellular fragments of NCAM and FAK to the nucleus. Studies by Westphal et al. reported that clustering of NCAM in hippocampal of the midline.4 Additional studies showed that SynCAM-1 is expressed at neurites promotes outgrowth by inducing Caspase-8 and -3-dependent the cell surface of growth cones and, in concert with focal adhesion kinase modifi cation of the submembranous Spectrin meshwork.12 (FAK), regulates growth cone morphology and axodendritic contact with target neurites.5 Transgenic experiments also suggest that SynCAM-1 The L1CAM group of proteins, which includes NCAM-L1, Neurofascin, Nr- specifi cally promotes the formation of excitatory synapses and can aff ect CAM, and CHL-1, have been shown to exert a broad range of adhesion- the plasticity of mature synaptic connections.6 Recent reports revealed dependent neural eff ects. The hippocampus-specifi c deletion of mouse that post-translational modifi cation is an important factor governing NCAM-L1 enhanced basal excitatory transmission, suggesting a role SynCAM biology. For example, N-glycosylation of the Ig1 domain for NCAM-L1 at inhibitory synapses.13 Neurofascin, which is known to enhances SynCAM-1 mediated synaptic adhesion, but has the opposite mediate myelination, may be a target for autoantibodies during Guillian- eff ect on SynCAM-2.7 Barré syndrome-induced destruction of the peripheral nervous system.14 Interactions between NrCAM and Neuropilin-2 are required for Sema- phorin 3F dependent guidance of thalamocortical neurons, disruption of which is believed to cause autism spectrum disorders.15 In the subventricular zone (SVZ) of mouse brain, knockout of CHL-1 increased the Postsynaptic number of neural progenitor cells and promoted neuronal diff erentiation, suggesting the physiological functions of CHL-1 include negatively regulating these processes.16 NCAM 120 TrkB References

1. Park, J. et al. (2008) J. Neurosci. 28:12815.

NCAM 140/180 2. Spiegel, I. et al. (2007) Nat. Neurosci. 10:861. 3. Biederer, T. & M. Stagi (2008) Curr. Opin. Neurobiol. 18:261. BDNF 4. Niederkofl er, V. et al. (2010) Development 137:427.

Presynaptic 5. Stagi, M. et al. (2010) Proc. Natl. Acad. Sci. USA 107:7568. 6. Robbins, E.M. et al. (2010) Neuron 68:894. FAK Fyn 7. Fogel, A.I. et al. (2010) J. Biol. Chem. 285:34864. Caspase-3 Caspase-8 8. Dityatev, A. et al. (2004) J. Neurosci. 24:9372. E-Spectrin D-Spectrin PKA 9. Kulahin, N. et al. (2011) FEBS Lett. 585:58. NCAM C-term ERK1 10. Cassens, C. et al. (2010) J. Biol. Chem. 285:28959. FAK N-term Nucleus 11. Kleene, R. et al. (2010) J. Neurosci. 30:10784. 12. Westphal, D. et al. (2010) J. Biol. Chem. 285:42046.

13. Law, J.W. et al. (2003) J. Neurosci. 23:10419. Domain Key: Ig-like Fibronectin type-III GPI-linked Cysteine-rich Leucine-rich Tyrosine kinase 14. Prüss, H. et al. (2011) Brain 134:e173.

N-Glycosylation Polysialic acid Phosphorylation site 15. Demyanerko, G.P. et al. (2011) J. Neurosci. 31:1545.

16. Huang, X. et al. (2011) Mol. Cell. Neurosci. 46:296. Figure 21. NCAM Interactions Promote Neurite Outgrowth. A summary of recent studies that investigated NCAM signaling during neurite outgrowth.9-12

34 For research use only. Not for use in diagnostic procedures. CELL ADHESION MOLECULES FOR NEUROSCIENCE

R&D Systems Products for SynCAM, NCAM, & L1CAM Family Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

IGSF4/SynCAM Family JAM-4/IGSF5 M (WB)

IGSF4A/SynCAM1 H M M (FC, WB) Kilon/NEGR1 HH (WB) M (WB)

IGSF4B/SynCAM3 HH (FC, IHC, WB) LAMP HH (B/N, WB)

IGSF4C/SynCAM4 HH (FC, IHC, WB) MAdCAM-1 H M H (FC) M (B/N, ELISA, IHC, WB) M

IGSF4D/SynCAM2 H MCAM/CD146 H (FC, IHC, WB) M (WB) R (FC, WB)

Cell Adhesion Molecules for Neuroscience MDGA1 HH (WB)

ALCAM/CD166 H M H (B/N, ELISA, FC, WB) M (FC, IHC, WB) H M MDGA2 HH (WB)

AMICA/JAML H M H (ELISA, FC, WB) M (WB) H MFG-E8 H M H (FC, WB) M (ELISA, WB) M

ASAM H NCAM-1/CD56 H M H (ELISA, FC, IHC, WB) M (WB) R (WB) H

BCAM HH (B/N, ELISA, WB) H NCAM-L1/L1CAM H M H (IHC, WB) M (FC)

BOC HH (WB) M (IHC, WB) Nephrin MH (IHC, WB) M (IHC, WB)

CD31/PECAM-1 H M P H (FC, IHC, IP, WB) M (FC, IHC, WB) R (FC) P (FC, WB) Neurofascin RH (IHC, WB) M (IHC, WB) R (IHC, WB)

CDO HH (FC, IHC, WB) M (IHC, WB) Ninjurin-1 H (WB)

CHL-1/L1CAM-2 H M H (IHC, WB) M (IHC, WB) Ninjurin-2 H (IHC, WB)

CLP24 H (FC, IHC) NrCAM HH (ELISA, IHC, WB) H

DSCAM HH (FC, IHC, WB) OBCAM/OPCML HH (FC, IHC, WB)

DSCAM-L1 HH (FC, WB) OCAM/NCAM2 M (WB) M

EpCAM/TROP-1 HH (ELISA, FC, IHC, WB) H PEAR1 HH (FC, WB)

ESAM HH (FC, IHC, WB) M (FC, IHC, WB) SALM2/LRFN1 HH (IHC, WB)

HepaCAM H (WB) SALM3/LRFN4 H

ICAM-1/CD54 H M R H (B/N, ELISA, FC, IHC, IP, WB) M (B/N, ELISA, FC, IHC, WB) H M R SALM4/LRFN3 HH (WB) R (B/N, ELISA, FC, IHC, WB) SIRPα/CD172a HH (FC, WB) ICAM-2/CD102 H M H (B/N, FC, WB) M (B/N, IHC, WB) TCAM-1 MM (WB) ICAM-3/CD50 HH (B/N, ELISA, FC, IHC, IP, WB) H Thrombospondin-1 HH (ELISA, IHC, IP, WB) H ICAM-5 H M H (IHC, WB) M (IHC, WB) TROP-2 HH (FC, IHC, WB) M (FC, IHC, WB) ISLR-2 H (WB) M (IHC, WB) UBE2S H JAM-A H M H (FC, IHC, WB) M (ELISA, IHC, WB) M VCAM-1/CD106 H M H (B/N, ELISA, FC, IHC, IP, WB) M (B/N, ELISA, FC, IHC, WB) H M JAM-B/VE-JAM H M H (B/N, WB) M (B/N, WB) VSIG3 H (WB) M (WB) JAM-C H M H (B/N, FC, WB) M (B/N, FC, IHC, WB)

120

A. B. 100

40 Relative Cell Number Cell Relative 20

0 100 101 102 103 104 IGSF4C/SynCAM4

Neurofascin-induced Neurite Outgrowth. Rat cortical neurons were cultured Detection of IGSF4C/SynCAM4 by Flow Cytometry. The A172 human glioblastoma NCAM-1/CD56 in Human Brain. Neural Cell Adhesion Molecule 1 (NCAM-1)/CD56 on a microplate under control conditions (A), or in the presence of immobilized cell line was stained with a Goat Anti-Human IGSF4C/SynCAM4 Antigen was detected in immersion-fi xed paraffi n-embedded sections of human brain using Recombinant Rat Neurofascin (Catalog # 3235-NF; B). The presence of Neurofascin Affi nity-purifi ed Polyclonal Antibody (Catalog # AF4164, fi lled histogram) or a Goat a Mouse Anti-Human NCAM-1/CD56 Monoclonal Antibody (Catalog # MAB24081). signifi cantly enhanced neurite outgrowth. IgG Isotype Control Antibody (Catalog # AB-108-C, open histogram), followed by a The tissue was stained using the Anti-Mouse HRP-DAB Cell & Tissue Staining Kit PE-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # F0107). (Catalog # CTS002; brown) and nuclei were counterstained with hematoxylin (blue).

www.RnDSystems.com/go/CellAdhesion 35 NEURAL STEM CELLS

Neural Stem Cells & Diﬀ erentiation Markers Neural stem cells (NSC) are undiff erentiated precursor cells defi ned by are specifi cally located at the subventricular zone (SVZ) in the developing their capacity for self-renewal and multipotency. Through proliferation telencephalon. In the adult brain, adult progenitors in the subgranular and division, NSCs generate clonally related progeny that diff erentiate to zone (SGZ) of the dentate gyrus in the hippocampus and the SVZ of the form all the major cell types of the CNS (Figure 22). These cells include lateral ventricles, are thought to functionally contribute to brain plasticity neurons, astrocytes, oligodendrocytes, and the ependymal cells that line and repair.2 the ventricles. The symmetric division of NSCs underlies their ability to self- Research has shown that Notch signaling is critical for NSC proliferation renew and serves to maintain the NSC population. In contrast, asymmetric and maintenance of the NSC phenotype. In the embryo, Notch signaling mitosis produces one NSC and one neural progenitor cell (NPC), daughter pathways maintain the NSC population by inhibiting neuronal diff er- cells with restricted diff erentiation capacity for neuronal or glial lineages. entiation.3 Moreover, ablation of Notch-1 during embryogenesis induces In addition, terminal asymmetric division generates two NPCs, but does the premature diff erentiation of NSC to radial glial cells, supporting the not contribute to maintaining the NSC pool. Extrinsic factors believed to hypothesis that Notch signaling controls lineage determination.4 Recent be essential for NSC maintenance and proliferation include Epidermal studies suggest that antagonism of the Notch pathway is essential for Growth Factor (EGF), Fibroblast Growth Factors, Sonic Hedgehog (Shh), controlling the balance between self-renewal and neurogenesis.5,6 Aguirre and members of the Wnt family. et al. studied the relationship between NSCs and NPCs in the mouse SVZ. There are four types of NPC.1 Neurogenesis in mammals commences Their fi ndings indicated that EGF receptor (EGF R)-mediated suppression with the induction of the neuroectoderm, followed by the formation of of Notch signaling caused expansion of the NPC pool and reduced NSC the neural plate, which folds to form the neural tube. These structures are number, an eff ect that was non-cell autonomous and dependent on cell- composed of neuroepithelial progenitors (NEP) that are responsible for cell contact.7 EGF R is known to be important for the proliferation and neurogenesis in the neural tube. NEPs also give rise to two other types migration of NPCs.8 Studies by Scott et al. reported that Shh was required of NPC, radial glia and basal progenitors. Radial glia are the dominant to maintain the multipotency of NSCs, an action that was dependent progenitor cell type in the developing brain, whereas basal progenitors on the transcription factor SOX9.9 Additional studies of NPCs suggested that an interaction between the Notch and Shh pathways controls the switch from symmetric proliferative division of NPCs to neurogenic Neural Stem Cells diff erentiation.10 CELL SURFACE: ABCG2 CD133 CXCR4 FGF R4 Frizzled-9 Glut1 SSEA-1 Notch-1 & -2 INTRACELLULAR: BMI-1 Brg1 FABP7 ASCL1/Mash1 Musashi-1 & -2 Nestin NeuroD1 Nucleostemin SOX1, 2, & 9 Vimentin Elucidation of the signaling pathways that govern NSC biology may have important implications for the treatment of brain cancers and neuro- SELF-RENEWAL degenerative diseases. For example, recent studies suggest that Insulin- Neural Progenitor Cells like Growth Factor II (IGF-II) stimulates the proliferation of NPCs and that RADIAL GLIAL CELLS: BLBP/FABP7 GLAST/SLC1A3 GFAP IGF-II levels were elevated in the cerebrospinal fl uid of patients with Nestin RC1&2 S100B Vimentin Glial-Restricted BASAL PROGENITORS: PAX6– SOX2– TBR2/EDMES Progenitors glioblastoma multiforme.11 In contrast, harnessing the proliferative po- A2B5 tential of NSCs provides hope for the development of novel therapeutic FGF Receptors Nestin strategies to combat neuronal loss induced by neurodegenerative Motor Neuron disease. New modes of treatment may exploit NSC pathotropism, the Progenitors migration and honing of NSCs to sites of neuronal injury, to enable the Neuron-Restricted Islet-1 & -2 Type 1 Astrocytes Progenitors Lhx3 CD44 delivery of gene therapies. Specifi cally, NSCs are attracted by factors that FGF R3 Doublecortin Neurogenin-2 GFAP are released during physiological events that occur in response to brain NCAM Olig2 Glast MAP2 Ran-2 pathology including infl ammation (MCP-1), reactive astrogliosis (SDF-1, E-III Tubulin Oligodendrocyte-Type 2 S100B CXCL12), and angiogenesis (SDF-1, VEGF).12 Astrocyte (0-2A) Precursors A2B5 References Nestin NG2/MCSP 1. Conti, L. & E. Cattaneo (2010) Nat. Rev. Neurosci. 11:176. Olig1 & 2 PDGF RD Differentiated 2. Deng, W. et al. (2010) Nat. Rev. Neurosci. 11:339. Post-Mitotic Neuronal Cells 3. Gaiano, N. & G. Fishell (2002) Annu. Rev. Neurosci. 25:471. NeuN Motor Neurons Islet-1 & -2 Neurofilaments (e.g. NF-L, NF-M) Type 2 Astrocytes 4. Gaiano, N. et al. (2000) Neuron 26:395. Lhx3 Neurotransmitter Synthesizing A2B5 Enzymes (e.g. GAD, TH) Neurogenin-2 CD44 5. Hoeck, J.D. et al. (2010) Nat. Neurosci. 13:1365. Synaptic Proteins Olig2 GFAP (PSD-95, Synaptophysin, VAMP) Glast 6. Kaltezioti, V. et al. (2010) PLoS Biol. 8:e1000565. ZENON Oligodendrocytes Musashi-1 GalC S100B 7. Aguirre, A. et al. (2010) Nature 467:323. MOG Myelination Antigen 8. Lillien, L. & H. Raphael (2000) Development 127:4993. O1 O4 9. Scott, C.E. et al. (2010) Nat. Neurosci. 13:1181. SOX10 10. Dave, R.K. et al. (2011) PLoS One 6:e14680.

11. Lehtinen, M.K. et al. (2011) Neuron 69:893.

Figure 22. Neural Stem Cell Diﬀ erentiation Markers. Illustration depicts the hierarchy of neural stem cell diff erentiation. 12. Müller, F.J. et al. (2006) Nat. Rev. Neurosci. 7:75.

36 For research use only. Not for use in diagnostic procedures. NEURAL STEM CELLS

R&D Systems Products for Neural Stem Cell Expansion & Diﬀ erentiation RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Neural Stem Cell & Progenitor Markers SOX21 H (IHC, WB)

ABCG2 H (FC, IHC) SOX9 H (IHC, WB)

ASCL1/Mash1 M (IHC, WB) SSEA-1 H (FC, IHC, IP) M (FC, IHC, IP)

BMI-1 H (ChIP, FC, IHC, WB) H Vimentin HH (FC, IHC, WB)

Brg1 H (WB) Neuronal Lineage Markers

CDCP1 H (FC, IHC, IP, WB) M (FC, IHC, WB) ACE/CD143 H M H (FC, IHC, IP, WB) M (ELISA, FC, IHC, IP, WB) H M

CXCR4 H (B/N, FC, IHC) M (B/N, FC, IHC) F (FC, IHC) ALCAM/CD166 H M H M

FABP7/B-FABP H (IHC, WB) -Internexin H (IHC, WB) M (WB) R (WB)

FGF R4 HH (FC, IHC, WB) M (IHC, WB) H CD90/Thy1 H (FC)

Frizzled-9 M (IHC, WB) GAD1/GAD67 H (IHC, WB)

GFAP H (IHC, WB) GAD2/GAD65 H (IHC, WB)

Glut1 H (FC, IHC) Glut1 H (FC, IHC)

HOXB1 H (IHC) HOXA1 H (IHC, WB)

Musashi-1 H (IHC, WB) Latexin H M H (IP, WB) M (B/N, IP, WB)

Musashi-2 H (WB) MSX1 H (WB) M (WB)

Nestin H (FC, IHC) M (FC, IHC, WB) R (FC, IHC, WB) NCAM-L1/L1CAM H M H (IHC, WB) M (FC)

NeuroD1 H (IHC, WB) M (IHC, WB) Nectin-2/CD112 H M H (FC, WB) M (WB)

Noggin H M M (IHC, WB) NeuroD1 H (IHC, WB) M (IHC, WB)

Notch-1 H M R H (ChIP, ELISA, FC, IHC, WB) M (FC, IHC, WB) H NeuroD2 H (WB) M (WB) R (B/N, FC, IHC, WB) NF-H H (IHC, WB) Notch-2 H M R H (FC, IHC, WB) M (FC, WB) R (B/N, FC, IHC, WB) NF-L H (IHC, WB) Nucleostemin H (IHC, WB) M (IHC, WB) R (IHC, WB) NF-M H (IHC, WB) Pax6 M (IHC) R (IHC) Ch (IHC) PRG-1/LPPR4 H (WB) PDGF R H M H (B/N, FC, IHC, IP, WB) M (B/N, IHC, WB) H M PSD-95 H (WB) M (WB) R (WB) Prominin 2 H (FC, IHC) ROBO3 H M H (IHC, WB) M (IHC, WB) S100B H (IHC, WB) Synaptophysin H (IHC, WB) R (IHC, WB) SOX1 H (IHC, WB) M (WB) TLE3 H (WB) M (WB) SOX2 H (ChIP, FC, IHC, WB) M (FC, IHC, WB) H M -III Tubulin Ms (FC, IHC, WB) Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot

-III Tubulin and Nestin in Diff erentiated Rat Cortical Stem Cells. -III Tubulin GFAP in Diff erentiated Rat Cortical Stem Cells. Glial Fibrillary Acidic Protein -III Tubulin, GFAP, and O4 in Diff erentiated Rat Cortical Stem Cells. III and Nestin were detected in immersion-fi xed Rat Cortical Stem Cells (Catalog # (GFAP) was detected in immersion-fi xed 7 day diff erentiated Rat Cortical Stem Tubulin, Glial Fibrillary Acidic Protein (GFAP), and Oligodendrocyte Marker O4 NSC001) using a Mouse Anti-Neuron-specifi c -III Tubulin Monoclonal (clone TuJ-1) Cells (Catalog # NSC001) using a NorthernLights-557-conjugated Sheep Anti- were simultaneously detected in immersion-fi xed 7 day diff erentiated Rat Cortical Antibody (Catalog # MAB1195) and a Goat Anti-Rat Nestin Antigen Affi nity-purifi ed Human GFAP Monoclonal Antibody (Catalog # NL2594R; yellow). Nuclei were Stem Cells (Catalog # NSC001) using the Human/Mouse/Rat Neural 3-Color Polyclonal Antibody (Catalog # AF2736). The cells were stained for -III Tubulin using counterstained with DAPI (blue). Immunocytochemistry Kit (Catalog # SC024). Proteins were detected using the NorthernLights™ 557-conjugated Donkey Anti-Mouse IgG Secondary Antibody antibodies included in the kit: a NorthernLights (NL) 637-conjugated mouse anti- (Catalog # NL007; red) and for Nestin using the NorthernLights 493-conjugated neuron-specifi c -III Tubulin monoclonal antibody (gray), a NL493-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL003; green). Nuclei were sheep anti-GFAP antigen affi nity-purifi ed polyclonal antibody (green), and a NL557- counterstained with DAPI (blue). conjugated mouse anti-O4 monoclonal antibody (red). Nuclei were counterstained with DAPI (blue).

www.RnDSystems.com/go/StemCells 37 NEURAL STEM CELLS

R&D Systems Products for Neural Stem Cell Expansion & Diﬀ erentiation RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Neuronal Lineage Markers, continued NGF R/TNFRSF16 H M H (FC, IHC, WB) M (IHC, WB)

Tyrosine Hydroxylase M (IF, IHC, WB) R (IF, IHC, WB) Ms (IF, IHC, IP, WB) NgR3/NgRH2 H (WB) Pr (IF, IHC, WB) Nogo Receptor/NgR H M H (B/N, WB) M (WB) VAMP-1 H (IHC, WB) M (IHC, WB) Olig 1, 2, 3 H (FC, IHC) VAMP-1/VAMP-2 H (IHC, WB) M (IHC, WB) Olig1 H (IHC, WB) M (IHC, WB) VAMP-2 H (IHC, WB) M (IHC, WB) R (WB) Olig2 H (ChIP, IHC, WB) H VAMP-7 H (IHC, WB) Olig3 H (IHC, WB) M (IHC, WB) VAMP-8 H (WB) Oligodendrocyte H (FC, IHC) M (FC, IHC) R (FC, IHC) Ch (FC, IHC) Motor Neuronal Markers Marker O1

Islet-1 H (IHC, WB) Oligodendrocyte H (FC, IHC) M (FC, IHC) R (FC, IHC) Ch (FC, IHC) Marker O4 Islet-2 H (IHC, WB) OMgp H M H (WB) M (WB) Neurogenin-2 H (IHC) R (IHC) PDGF R H M H (B/N, FC, IHC, IP, WB) M (B/N, IHC, WB) H M Olig2 H (ChIP, IHC, WB) H PLP H (IHC) Glial Lineage Markers S100B H (IHC, WB) A2B5 H (FC, IHC) M (FC, IHC) R (FC, IHC) Ch (FC, IHC) SLC22A1 H (WB) Carbonic Anhydrase HH (IHC, IP, WB) II/CA2 SOX10 H (IHC, WB) R (IHC)

CD44 H M R H (FC, IHC, IP, WB) M (B/N, WB) R (B/N, WB) Ca (FC) TROY/TNFRSF19 H M H (WB) M (ELISA, IHC, WB) M Claudin-11 H (FC) Neural Crest Cell Markers

Claudin-12 H (FC, IHC) ASCL1/Mash1 M (IHC, WB)

FABP6 H (IHC, WB) BMP-4 H M Z H (B/N, ELISA, IHC, WB) Z (WB) H

FGF R1-4 H (WB) BMP-7 H M H (B/N, ELISA, IHC, WB) H

FGF R1 HH (B/N, WB) H Integrin 4/CD49d H (B/N, FC, IHC) M (FC, WB) FGF R1 H Integrin 41 H M FGF R1 H Integrin 47/ H LPAM-1 FGF R2 H M H (B/N, FC, IHC, WB) M (B/N, WB) H Neurogenin-1 H (WB) FGF R2 H M H (WB) H Neurogenin-2 H (IHC) R (IHC) FGF R2 H M Neurogenin-3 H (WB) FGF R3 H M H (B/N, FC, IHC, WB) M (B/N) H NGF R/TNFRSF16 H M H (FC, IHC, WB) M (IHC, WB) FGF R4 HH (FC, IHC, WB) M (IHC, WB) H Notch-1 H M R H (ChIP, ELISA, FC, IHC, WB) M (FC, IHC, WB) H FGF R5/FGFRL1 MH (WB) M (IHC, WB) R (B/N, FC, IHC, WB)

GFAP H (IHC, WB) Notch-2 H M R H (FC, IHC, WB) M (FC, WB) R (B/N, FC, IHC, WB)

GMF- H (IHC, WB) Numb H (IHC, WB) H M R

LINGO-1 H (FC, IHC, WB) Pax3 H (FC, IHC, WB) M (FC, IHC, WB)

LRRN1/NLRR-1 H (IHC, WB) M (IHC, WB) Snail H (ChIP, IHC, WB) H

LRRN3/NLRR-3 H SOX10 H (IHC, WB) R (IHC) MAG/Siglec-4a RR (B/N, ELISA, IHC, WB) R Neural Stem Cell Growth Factors & Receptors

MBP H (WB) M (WB) R (WB) B (WB) Macroglobulin HH (IHC, IP, WB) M (WB)

Meteorin MM (ELISA, IHC, WB) M Activin A H M R H (B/N, ELISA, IHC, WB) M (B/N, ELISA, IHC, WB) H M R Meteorin-like/ M R (B/N, ELISA, IHC, WB) METRNL Activin AB H MOG H (IHC, WB) M (IHC, WB) Activin AC H

Musashi-1 H (IHC, WB) Activin B HH (B/N, WB)

Nestin H (FC, IHC) M (FC, IHC, WB) R (FC, IHC, WB) Activin C H (IHC, WB) M (WB)

Neurofascin RH (IHC, WB) M (IHC, WB) R (IHC, WB) Activin RIA/ALK-2 H M H (FC, IHC, WB)

Neuroglycan C/ H M H (IHC, WB) M (IHC, WB) R (IHC, WB) Activin RIB/ALK-4 H M H (B/N, FC, WB) M (B/N, IHC, WB) CSPG5 Activin RIIA H M H (B/N, FC, IHC, WB) NG2/MCSP H (FC, IHC, IP, WB) M (FC, IHC) R (FC)

38 For research use only. Not for use in diagnostic procedures. NEURAL STEM CELLS

R&D Systems Products for Neural Stem Cell Expansion & Diﬀ erentiation RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Neural Stem Cell Growth Factors & Receptors, continued EGF H M R H (B/N, ELISA, IHC, WB) M (B/N, ELISA, IHC, WB) H M R R (B/N, ELISA, WB) Activin RIIA/B H (WB) EGF R/ErbB1 H M H (ELISA, FC, IHC, IP, WB) M (IHC, WB) H Activin RIIB H M H (B/N, FC, IHC, WB) EGF-L6 M BMP-1/PCP HH (IHC, IP, WB) FGF acidic H M B H (B/N, IHC, WB) M (WB) B (B/N, WB) H BMP-10 H M H (B/N, WB) M (B/N, IHC, WB) FGF basic H M R B H (B/N, ELISA, IHC, WB) B (B/N, WB) H BMP-15/GDF-9B HH (IHC, WB) M (IHC, WB) R (WB) FGF R1 HH (B/N, WB) H BMP-2 H Z H (B/N, ELISA, IHC, WB) Z (B/ N, WB) H M R FGF R1-4 H (WB) BMP-2/BMP-4 H (B/N, IHC, WB) Z (WB) FGF R1 H BMP-2/BMP-7 HH (WB) Heterodimer FGF R1 H

BMP-2a Z FGF R2 H M H (B/N, FC, IHC, WB) M (B/N, WB) H

BMP-3 HH (IHC, WB) FGF R2 H M H (WB) H

BMP-3b/GDF-10 HH (IHC, WB) FGF R2 H M

BMP-4 H M Z H (B/N, ELISA, IHC, WB) Z (WB) H FGF R3 H M H (B/N, FC, IHC, WB) M (B/N) H

BMP-4/BMP-7 H FGF R4 HH (FC, IHC, WB) M (IHC, WB) H Heterodimer FGF R5/FGFRL1 MH (WB) M (IHC, WB) BMP-5 H M H (B/N, ELISA, IHC, WB) M (IHC, WB) H FGF-10 H M H (IHC, WB) M (IHC, WB) BMP-6 H M H (B/N, ELISA, IHC, WB) M (IHC, WB) H FGF-11 H (WB) BMP-7 H M H (B/N, ELISA, IHC, WB) H FGF-12 HH (WB) BMP-8 H (IHC, WB) FGF-13 H (WB) BMP-8a H FGF-15 M (IHC) BMP-8b H (WB) M (WB) FGF-16 HH (B/N, WB) BMP-9 H M H (B/N, ELISA, WB) M (B/N, ELISA, WB) H FGF-17 HH (B/N, IHC, WB) BMPR-IA/ALK-3 H M H (FC, IHC, WB) FGF-19 HH (B/N, ELISA, IHC, WB) H BMPR-IB/ALK-6 H M H (FC, IHC, WB) M (WB) FGF-20 HH (B/N, IHC, WB) BMPR-II H M H (FC, IHC, WB) FGF-21 HH (FC, IHC, WB) M (WB) H M CELSR2 H (IHC, WB) FGF-22 HH (B/N, WB) Desert Hedgehog/ H M M (IHC, WB) Dhh FGF-23 H M H (B/N, WB) M (IHC, WB)

Draxin H M H (IHC, WB) M (IHC, WB) R (IHC, WB) FGF-3 HH (B/N, IHC, WB)

Oligodendrocyte Marker O4 in Diff erentiated Rat Cortical Stem Cells. Olig2 and Oligodendrocyte Marker O4 in Diff erentiated Rat Cortical Stem Cells. Nestin in Undiff erentiated Rat Cortical Stem Cells. Nestin was detected in Oligodendrocyte Marker O4 was detected in immersion-fi xed 7 day diff erentiated Olig2 and Oligodendrocyte Marker O4 were detected in immersion-fi xed 7 day immersion-fi xed undiff erentiated Rat Cortical Stem Cells (Catalog # NSC001) using Rat Cortical Stem Cells (Catalog # NSC001) using a Mouse Anti-Human/Mouse/Rat/ diff erentiated Rat Cortical Stem Cells (Catalog # NSC001) using Goat Anti-Human a Mouse Anti-Rat Nestin Monoclonal Antibody (Catalog # MAB2736). The cells Chicken Oligodendrocyte Marker O4 Monoclonal Antibody (Catalog # MAB1326). The Olig2 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF2418) and a Mouse were stained using the NorthernLights 557-conjugated Donkey Anti-Mouse IgG cells were stained using an anti-mouse IgM secondary antibody (green) and nuclei Anti-Human/Mouse/Rat/Chicken Oligodendrocyte Marker O4 Monoclonal Antibody Secondary Antibody (Catalog # NL007; red) and counterstained with DAPI (blue). were counterstained with DAPI (blue). (Catalog # MAB1326). The cells were stained for Olig2 using the NorthernLights™ 637-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL002; red), and stained for O4 using an anti-mouse IgM secondary antibody (pseudo-stained green). Nuclei were counterstained with DAPI (blue).

www.RnDSystems.com/go/StemCells 39 NEURAL STEM CELLS

R&D Systems Products for Neural Stem Cell Expansion & Diﬀ erentiation RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Neural Stem Cell Growth Factors & Receptors, continued IGF-I R H M H (B/N, ELISA, FC, IHC, WB) M (B/N, FC, IHC, WB) H

FGF-4 H M H (B/N, ELISA, IHC, WB) M (IHC, WB) H IGF-II H M H (B/N, IHC, WB) M (B/N, ELISA, IHC, WB) M

FGF-5 HH (B/N, IHC, WB) IGF-II R HH (B/N, ELISA, FC, IHC, WB) H

FGF-6 H M H (B/N, ELISA, WB) M (IHC) IGFL-3 H (IHC, WB)

FGF-8 H M H (B/N, IHC, WB) M (B/N, IHC, WB) Indian Hedgehog/ H M H (WB) M (IHC, WB) Ihh FGF-9 HH (B/N, ELISA, IHC, WB) H KGF/FGF-7 H M Ca H (B/N, ELISA, IHC, WB) Ca (IHC) H FGF-BP H R H (WB) R (B/N, IHC, WB) LRP-1 Cluster II HH (WB) Fibronectin H B H (FC, IHC, IP, WB) LRP-1 Cluster III HH (IHC, WB) Frizzled-1 H M H (FC, IHC, WB) M (FC, IHC, WB) LRP-1 Cluster IV H Frizzled-10 HH (FC, IHC, IP, WB) LRP-1B H (WB) Frizzled-2 MM (WB) LRP-4 H (IHC, WB) R (IHC, WB) Frizzled-3 H (FC, IHC, WB) M (FC, IHC, WB) LRP-5 H (WB) Frizzled-4 H M H (FC, IHC, WB) M (FC, IHC, WB) LRP-6 H M H (FC, IHC, WB) M (WB) Frizzled-5 HH (WB) MESDC2 MH (IHC, WB) M (IHC, WB) Frizzled-6 H (FC, WB) M (FC, IHC, WB) Meteorin MM (ELISA, IHC, WB) M Frizzled-7 H M H (FC, IHC) M (FC, IHC, WB) Meteorin-like/ M Frizzled-8 H M M (IHC, WB) METRNL

Frizzled-9 M (IHC, WB) NGF R/TNFRSF16 H M H (FC, IHC, WB) M (IHC, WB)

GDF-1 HM (WB) Noggin H M M (IHC, WB)

GDF-11/BMP-11 H Patched 1/PTCH M (FC, IHC, WB)

GDF-15 HH (ELISA, WB) M (IHC) H Patched 2/PTCH2 H (FC, IHC, WB)

GDF-3 H M H (IHC, WB) M (IHC, WB) PDGF H P H (B/N, WB) Ms (B/N, WB)

GDF-5/BMP-14 MM (B/N, IHC, WB) PDGF R H M H (B/N, FC, IHC, IP, WB) M (B/N, IHC, WB) H M

GDF-6/BMP-13 M PDGF R H M H (B/N, FC, IHC, IP, WB) M (IHC, WB) H M

GDF-7/BMP-12 MM (WB) PDGF-A H (ELISA, WB)

GDF-8/Myostatin H M R H (B/N, IHC, WB) M (B/N, IHC, WB) R (B/N, IHC, WB) PDGF-AA H R H (B/N, ELISA, IHC, WB) R (B/N, IHC, WB) Ms (B/N, WB) H M

GDF-9 MM (IHC, WB) PDGF-AB H R H (B/N, ELISA, IHC, WB) Ms (B/N, WB) H M R

GFR-1/GDNF R-1 H R H (IHC, WB) R (B/N, IHC, WB) PDGF-B H (B/N, ELISA, WB) Ms (B/N, WB)

GFR-2/GDNF R-2 H M H (B/N, IHC, WB) M (B/N, IHC, WB) PDGF-BB H R H (B/N, ELISA, WB) H M R

GFR-3/GDNF R-3 H M H (IHC, WB) M (IHC, WB) PDGF-C H (B/N, IHC, WB) M (B/N, IHC, WB) GFR-4/GDNF R-4 H (WB) M (IHC, WB) PDGF-CC H M

IGFBP-1 H M H (B/N, ELISA, WB) M (IHC, WB) H PDGF-D H (B/N, IHC, WB) IGFBP-2 H M H (B/N, ELISA, WB) M (B/N, ELISA, WB) H M PDGF-DD H

IGFBP-3 H M H (B/N, ELISA, IHC, WB) M (ELISA, WB) H M PlGF-2 MM (B/N, ELISA, WB) M

IGFBP-4 HH (B/N, ELISA, IHC, WB) H ROR1 Receptor H (FC, WB) H Tyrosine Kinase IGFBP-5 H M H (ELISA, IHC, WB) M (ELISA, IHC, WB) H M RTK-like Orphan H (FC, WB) H IGFBP-6 H M H (B/N, ELISA, WB) M (ELISA, IHC, WB) H M Receptor 2/ROR2 IGFBP-L1 MH (WB) M (IHC, WB) Ryk H (WB) M (IHC, WB) IGFBP-rp1/IGFBP-7 H M H (IHC, WB) M (IHC, WB) SDNSF/MCFD2 H (IHC, WB) M (IHC, WB) IGFBP-rP10 H (WB) M (WB) Sonic Hedgehog/Shh H M H (FC, WB) M (B/N, ELISA, FC, IHC, WB) M IGF-I H M R H (B/N, ELISA, IHC, WB) M (B/N, ELISA, IHC, WB) H M R

40 For research use only. Not for use in diagnostic procedures. NEURAL STEM CELLS

R&D Systems Products for Neural Stem Cell Expansion & Diﬀ erentiation RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

TGF- HH (B/N, ELISA, IHC, WB) H Wnt-1 M (IHC, WB)

Neural Stem Cell Growth Factors & Receptors, continued Wnt-2 H (IHC, WB)

TGF- RI/ALK-5 MH (FC, IHC, WB) M (FC, WB) Wnt-2b M (IHC, WB)

TGF- RII H M H (B/N, ELISA, FC, IHC, WB) M (FC, WB) H Wnt-3a H M H (B/N, WB) M (B/N, WB)

TGF- RIIb HH (WB) Wnt-4 H M H (WB) M (IHC, WB)

TGF- RIII H M H (ELISA, FC, WB) M (FC, WB) H Wnt-5a H M H (IHC, WB) M (IHC, WB) R (IHC, WB)

VEGF H M R Ca F Z H (B/N, ELISA, FC, IF, IHC, WB) M (B/N, ELISA, IHC, WB) H M R Ca Wnt-5b MM (IHC) R (B/N, ELISA, IHC, WB) Ca (B/N, ELISA, IHC, WB) Z (B/N, WB) Wnt-6 H (IHC, WB) VEGF R1, R2, R3 H (FC) Wnt-7a HH (IHC, WB) VEGF R1/Flt-1 H M H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M Wnt-7b H (IHC, WB) VEGF R2/KDR/Flk-1 H M H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M Wnt-8a M (IHC, WB) VEGF R3/Flt-4 H M H (ELISA, FC, IHC, WB) M (ELISA, FC, WB) H M Wnt-8b H (IHC, WB) M (IHC, WB) VEGF/PlGF HH (WB) H Heterodimer Wnt-9a H (IHC, WB)

VEGF-B H M H (IHC, WB) M (B/N, IHC, WB) Wnt-9b MH (IHC, WB) M (IHC, WB)

VEGF-C HH (IHC, WB) M (WB) R (WB) H Wnt-10b MM (WB)

VEGF-D H M H (B/N, ELISA, IHC, WB) M (ELISA, IHC, WB) H M Wnt-11 HH (IHC, WB) M (IHC, WB)

Neural Stem Cells: Primary Cells, Media, Supplement & Complete Kits

PRODUCT KIT CONTENTS/DESCRIPTION CATALOG # SIZE Primary Cortical Stem Cells Rat Cortical Stem Cells 1 vial; Vial contains 3 x 106 cells NSC001 1 vial

Mouse Cortical Stem Cells 2 vials; Each vial contains 2 x 106 cells NSC002 2 vials N-2 MAX Media Supplement N-2 MAX Media Supplement (100X) A modifi cation of Bottenstein’s formulation, providing optimal conditions for neural stem cell expansion. Suffi cient for 500 mL of medium. AR009 5 mL Neural Progenitor Cell Marker Kit Human/Mouse/Rat Neural Progenitor Contains 25 g each of antibodies to rat Notch-2, human CXCR4, human Vimentin, human/mouse SSEA-1, human Musashi-1, SC025 1 Kit Cell Marker Antibody Panel human SOX1, human/mouse SOX2, and rat Nestin Neural Precursor Cell-based Screening & Bioassay Kit Neural Precursor Cell-based Neural Stem Cell Maintenance Supplement, Neural Diff erentiation Supplement, Fibronectin, HRP-conjugated Anti-Neuron-Specifi c SC014 1 Kit Screening & Bioassay Kit -III Tubulin, Resazurin, Buff ers, Substrates, and Diluents Neural Diﬀ erentiation Kits Human/Mouse Dopaminergic ITS Media Supplement, N-2 Plus Media Supplement, FGF basic, FGF-8b, Fibronectin, Shh-N SC001B 1 Kit Neuron Diff erentiation Kit Mouse Oligodendrocyte ITS Media Supplement, N-2 Plus Media Supplement, EGF, FGF basic, Fibronectin, PDGF-AA SC004 1 Kit Diff erentiation Kit Neural Multi-Color Immunocytochemistry Kit Human/Mouse/Rat Neural 3-Color NL557-conjugated Anti-Oligodendrocyte Marker O4, NL637-conjugated Anti--III Tubulin, NL493-conjugated Anti-GFAP SC024 1 Kit Immunocytochemistry Kit Fibronectin Coated Microplates Human Fibronectin Coated 96-well Clear, polystyrene tissue culture microplates coated with 0.2 micron fi ltered serum-derived human fi bronectin and/or blocked CWP001 5 pack Microplates with Bovine Serum Albumin Fraction V under aseptic conditions Bovine Fibronectin Coated 96-well Clear, polystyrene tissue culture microplates coated with 0.2 micron fi ltered serum derived bovine fi bronectin and/or blocked CWP002 5 pack Microplates with Bovine Serum Albumin Fraction V under aseptic conditions

For more details, please see Specialized Tools page 96.

www.RnDSystems.com/go/StemCells 41 NEURAL STEM CELLS

Adult Neurogenesis Ramon y Cajal’s contention that the adult mammalian brain was incapable of gene expression. In the unique environment of the mature nervous of generating new neurons was fi rst questioned by Altman et al. in the system, neurogenesis is regulated by both intrinsic and extrinsic factors. 1960s, and by Kaplan and Hinds in the 1970s.1,2 The subsequent demon- Epigenetic mechanisms represent the major intrinsic infl uence on adult stration that neurogenesis was seasonally regulated in adult song bird neurogenesis. Epigenetics refers to changes in gene expression that brain evoked an explosion of research in the 1990s.3 Now it is established are independent of alterations to the genomic DNA sequence. This that adult neurogenesis, the generation of functional neuronal cells from may be achieved by DNA methylation, histone modifi cation, chromatin adult neural progenitors, occurs continuously in two distinct regions of remodeling, and/or transcriptional feedback loops.7 the mammalian brain. Adult neurogenesis is also aff ected by extrinsic factors including a In the subgranular zone (SGZ) of the dentate gyrus in the hippocampus broad range of physiological, pharmacological, and environmental adult neural stem cells diff erentiate into dentate granule cells. Following stimuli. Extracellular growth factors, hormones, and neurotransmitters a lengthy period of maturation, these cells integrate into the pre-existing have been shown to promote neurogenesis. The most studied of these neural circuitry and are believed to functionally contribute to the pro- factors are BDNF, FGF, and VEGF. BDNF has been shown to induce neural cesses of learning and memory (Figure 23).4 In the subventricular zone progenitor cell mitosis in the SGZ, and enhance the migration and survival (SVZ) of the lateral ventricles, adult neural stem cells form neuroblasts and of neuroblasts in the SVZ.8-10 FGF basic (FGF-2) is a powerful mitogen for a small number (~5%) of glia.5 The neuroblasts move through the rostral hippocampal neural stem progenitor cells, and FGF-15 was shown to be migratory stream (RMS) to the olfactory bulb where they diff erentiate essential for initiation and progression of neurogenesis in the midbrain.11,12 into granule cells and periglomerular cells that function in olfaction. It VEGF was reported to promote neural progenitor cell migration along the was estimated that up to fi fty thousand new neurons are generated each RMS via VEGF R1 and increase neurogenesis following traumatic brain day in the rat SVZ, but only 40% of these cells survive throughout the injury through VEGF R2.13,14 lifetime of the animal.6 Nearly all the cells that diff erentiate into neuronal Consistent with a growing number of reports that neurotransmitters can phenotypes become GABA or Calretinin positive inhibitory interneurons, act as trophic factors, neurotransmitters have been shown to enhance the with a smaller percentage forming tyrosine hydroxylase positive diff erentiation and survival of adult-born neurons.15 For example, GABA dopaminergic neurons. is thought to be essential for the integration of new neurons, and loss Cell proliferation and diff erentiation during adult neurogenesis is of dopaminergic input reduces progenitor proliferation in the SVZ.16,17 In dependent on the tightly orchestrated spatial and temporal control contrast, stress hormones and corticosteroids have been shown to negatively impact adult neurogenesis.18 Similarly, pro-infl ammatory Week 0: Proliferation & Fate Determination cytokines sup press adult neurogenesis following activation of microglia. PP However, microglia also exert neurogenic eff ects following stimulation by ML TGF-, supporting a pro-neurogenic role for anti-infl ammatory cytokines in the adult brain.19 GCL References

1. Altman J. (1963) Anat. Rec. 145:573. NPC Adult-born DGC SGZ 2. Kaplan, M.S. & J.W. Hinds (1977) Science 197:1092. Week 1: Migration PP 3. Riddle, D.R. & R.J. Lichtenwainer (2007) Brain Aging: Models, Methods, and Mechanisms. CRC Press. ML 4. Deng, W. et al. (2010) Nat. Rev. Neurosci. 11:339.

GCL 5. Zhao, C. et al. (2008) Cell 132:645. 6. Winner, B. et al. (2002) Eur. J. Neurosci. :1681. GABAergic Interneuron 16 + SGZ GABA 7. Ma, D.K. et al. (2010) Nat. Neurosci. 13:1338.

Weeks 3-8: Maturation & Integration 8. Bath, K.G. & Lee, F.S. (2010) Dev. Neurobiol. 75:339. + Glutamate PP 9. Pinnock, S.B. et al. (2010) PLoS One 5:e13652. GABA − ML 10. Park, H.R. et al. (2010) Neurosci. Lett. 482:235.

11. Fischer, T. et al. (2011) Dev. Biol. 350:496. GCL 12. Rodrigo, C. et al. (2010) J. Neurochem. 113:615. GABA − SGZ 13. Wittko, I.M. et al. (2009) J. Neurosci. 29:8704. Mossy fibers to CA3 r 14. Lu, K.T. et al. (2011) J. Neurotrauma 28:441.

Figure 23. Adult Neurogenesis in the Hippocampus. Week 0: The process of adult neurogenesis is initiated by proliferation of 15. Pathania, M. et al. (2010) Neuropharmacology 58:865. neural progenitor cells (NPC) in the subgranular zone (SGZ) of the dentate gyrus and the initial diff erentiation into dentate granule 16. Sernagor, E. et al. (2010) Front. Cell. Neurosci. 4:11. cells (DGC).4 Week 1: Adult-born DGCs migrate into the granule cell layer (GCL), the dendrite extends toward the molecular layer (ML), and the axon projects in the direction of CA3. Immature DGCs receive excitatory GABAergic input from local interneurons. 17. Khaindrava, V. et al. (2011) Neurobiol. Dis. 42:284. Week 3-8: DGCs receive excitatory glutamatergic input from the perforant pathway (PP), GABAergic input becomes inhibitory, 18. Sahay, A. & R. Hen (2007) Nat. Neurosci. 10:1110. and eff erent and aff erent synapses are formed.By Week 8: Adult-born DGCs have undergone a lengthy period of maturation. At this point they are structurally and physiologically indistinguishable from mature DGCs, and are fully integrated within the 19. Mathieu, P. et al. (2010) Neuroimmunomodulation 17:200. pre-existing neural circuitry.

42 For research use only. Not for use in diagnostic procedures. NEURAL STEM CELLS

R&D Systems Products for Adult Neurogenesis RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Aldehyde HH (IHC, IP, WB) Histone Deacetylase H (IHC, WB) M (IHC, WB) R (IHC, WB) Dehydrogenase 4/HDAC4 1-A1/ALDH1A1 Histone Deacetylase HH (ChIP, WB) M (ChIP, WB) ASCL1/Mash1 M (IHC, WB) 8/HDAC8

BDNF HH (ELISA, FC, IHC, WB) H MBD3 H (WB)

BMI-1 H (ChIP, FC, IHC, WB) H MBD4 H (WB) M (WB)

Calbindin D H (IHC, WB) NCAM-1/CD56 H M H (ELISA, FC, IHC, WB) M (WB) R (WB) H

Calretinin H (IHC, WB) M (IHC, WB) R (IHC, WB) Nestin H (FC, IHC) M (FC, IHC, WB) R (FC, IHC, WB)

DNMT1 H (IHC, WB) NeuroD1 H (IHC, WB) M (IHC, WB)

DNMT3A H (IHC, WB) M (WB) NeuroD2 H (WB) M (WB)

EGF R/ErbB1 H M H (ELISA, FC, IHC, IP, WB) M (IHC, WB) H Neurogenin-2 H (IHC) R (IHC)

FABP7/B-FABP H (IHC, WB) Pax6 M (IHC) R (IHC) Ch (IHC)

FGF acidic H M B H (B/N, IHC, WB) M (WB) B (B/N, WB) H PDGF R H M H (B/N, FC, IHC, IP, WB) M (B/N, IHC, WB) H M

FGF basic H M R B H (B/N, ELISA, IHC, WB) B (B/N, WB) H SOX2 H (ChIP, FC, IHC, WB) M (FC, IHC, WB) H M

GADD45 H (WB) Tyrosine Hydroxylase M (IF, IHC, WB) R (IF, IHC, WB) Ms (IF, IHC, IP, WB) Pr (IF, IHC, WB) Galectin-1 H M H (FC, IHC, WB) M (ELISA, FC, IHC, WB) M VEGF H M R Ca F Z H (B/N, ELISA, FC, IF, IHC, WB) M (B/N, ELISA, IHC, WB) H M R Ca GFAP H (IHC, WB) R (B/N, ELISA, IHC, WB) Ca (B/N, ELISA, IHC, WB) Z (B/N, WB) Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot

A. 50 B. 120

40 100

80 30 60 20 40 Relative Cell Number Cell Relative Relative Cell Number Cell Relative 10 20

0 0 100 101 102 103 104 100 101 102 103 104 CXCR4 SSEA-1

Detection of Neural Cell Progenitor Markers CXCR4 and SSEA-1 by Flow Cytometry. Cells were stained with antibodies included in the Human/Mouse/Rat Neural SCF R/c-kit in Mouse Cerebellum. Stem Cell Factor Receptor (SCF R)/c-kit was Progenitor Cell Marker Antibody Panel* (Catalog # SC025). A. CXCR4 was detected in undiff erentiated Mouse Cortical Stem Cells (Catalog # NSC002) using a mouse anti- detected in perfusion-fi xed frozen sections of mouse brain using a Rat Anti-Mouse

CXCR4 monoclonal antibody (fi lled histogram) or a mouse IgG2A isotype control (open histogram). B. SSEA-1 was detected in undiff erentiated Rat Cortical Stem Cells (Catalog SCF R/c-kit Monoclonal Antibody (Catalog # MAB1356). The tissue was stained with # NSC001) using a mouse anti-SSEA-1 monoclonal antibody (fi lled histogram) or a mouse IgM isotype control (open histogram). Cells were stained using PE-conjugated the Anti-Rat HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS017; brown) and secondary developing reagents. *This panel also includes primary antibodies to detect Nestin, SOX1, SOX2, Vimentin, Notch-1, and Musashi-1. nuclei were counterstained with hematoxylin (blue). Specifi c labeling was localized to Bergmann glial cells located around Purkinje cells. FGF basic Antibody (Pg/mL) 10-2 10-1 100 101 102 30000 30000 Cell Proliferation (Mean CPM) 25000 25000

20000 20000

15000 15000

10000 10000

Cell Proliferation (Mean CPM) Proliferation Cell 5000 5000

0.0 0.0 10-3 10-2 10-1 100 101 FGF basic (ng/mL)

Notch-2 in Rat Choroid Plexus. Notch-2 was detected in perfusion-fi xed frozen Cell Proliferation Induced by FGF basic and Neutralization by a FGF basic GABAB R1 in Rat Trigeminal Ganglion. GABAB Receptor Subunit 1 (GABAB R1) was sections of rat brain using a Goat Anti-Rat Notch-2 Antigen Affi nity-purifi ed Antibody. Bovine FGF basic (Catalog # 133-FB) stimulates proliferation in the detected in perfusion-fi xed frozen sections of rat brain using a Sheep Anti-Mouse/

Polyclonal Antibody (Catalog # AF1190). The tissue was stained with the Anti-Goat NR6R-3T3 mouse fi broblast cell line in a dose-dependent manner (green line). Rat GABAB R1 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF7000). The HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS008; brown) and nuclei were Proliferation elicited by Bovine FGF basic (0.5 ng/mL) is neutralized (orange line) tissue was stained using the NorthernLights™ 557-conjugated Donkey Anti-Sheep counterstained with hematoxylin (blue). by increasing concentrations of a Rabbit Anti-Bovine FGF basic Antigen Affi nity- IgG Secondary Antibody (Catalog # NL010; red) and counterstained with DAPI (blue). purifi ed Polyclonal Antibody (Catalog # AB-33-NA). Specifi c staining was localized to the cell bodies of neurons. www.RnDSystems.com/go/AdultNeurogenesis 43 SYNAPTIC PROTEINS & NEUROTRANSMITTERS

Synaptic Proteins The human brain contains over one hundred trillion synapses, specialized zone.9 This process requires soluble N-ethylmaleimide-sensitive fusion intercellular junctions that facilitate the transfer of information from one protein attachment receptors (SNAREs), which catalyze fusion by forming neuron to another.1 Transmission of information across the synaptic cleft a complex that bridges the two fusing membranes.10 Calcium entry is fast, dynamic, highly effi cient, and tightly regulated. The formation induces simultaneous binding of Synaptotagmin to the vesicle and target of new synapses is prompted by the recognition of a target cell by the membranes, and assembly of the SNARE complex. Syntaxin-1 and SNAP- neuronal growth cone and is thought to involve four stages: initiation, 25 on the vesicle membrane, and VAMP/Synaptobrevin on the plasma specifi cation, diff erentiation, and maturation.2 Synapse formation and membrane, form an -helix that brings the two membranes together. function are dependent on trans-synaptic cell adhesion molecules that SM-proteins (Sec1/Munc18-like) bind to trans-SNARE complexes to direct bridge the synaptic cleft and mediate essential signaling between the fusion. Recently, Complexin has been shown to be an important part of presynaptic and postsynaptic specializations (Figure 24). the fusion machinery.11,12 Complexin binds SNARE complexes to both 13,14 Two of the most studied synaptic adhesion molecule families are suppress spontaneous fusion and promote calcium-evoked fusion. Neurexins and Neuroligins. Neurexins were discovered as receptors for References -Latrotoxin, a toxic component of black widow spider venom that binds 1. Eroglu, C. & B.C. Barres (2010) Nature 468:223. 3 to presynaptic receptors causing a massive release of neurotransmitter. 2. Südhof, T.C. (2008) Nature 455:903. Type I transmembrane proteins, Neurexins can be classifi ed as or ,   3 Ushkaryov, Y.A. et al. (2008) Handb. Exp. Pharmacol. 184:171. which diff er in size and extracellular amino acid sequence.2 Neuroligins are 4. Varoqueaux, F. et al. (2006) Neuron 51:741. ligands for Neurexins that are expressed at the postsynaptic density. Also type I transmembrane proteins, Neuroligins bind both  and  Neurexins. 5. Wang, P.Y. et al. (2008) Mol. Cell. Neurosci. 39:83. Although known to promote synaptogenesis in vitro, knockout ex- 6. Mah, W. et al. (2010) J. Neurosci. 30:5559. periments showed that the Neurexin-Neuroligin complex is critically 7. Chen, S.X. et al. (2010) Neuron 67:967. required for synaptic function but not formation.4 In contrast, members 8. Siddiqui, T.J. et al. (2010) J. Neurosci. 30:7495. of the Synaptic Adhesion-like Molecule (SALM) family, which are known 9. Pang, Z.P. & T.C. Südhof (2010) Curr. Opin. Cell. Biol. 22:496. to induce neurite outgrowth, are thought to regulate synaptogenesis.5,6 10. Paddock, B.E. et al. (2011) J. Neurosci. 31:2248. Recent studies suggest that Neurexin-Neuroligin interactions may promote synaptic dendritogenesis.7 In addition, Neurexin-1 was found 11. Hobson, R.J. (2011) Curr. Biol. 21:106. to bind LRRTM2 to induce presynaptic diff erentiation and postsynaptic 12. Yang, X. (2010) Neuron 68:907. assembly.8 13. Maximov, A. et al. (2009) Science 23:516. Synaptic transmission is dependent on calcium-triggered exocytosis of 14. Martin, J.A. et al. (2011) Curr. Biol. 21:97. synaptic vesicles packed with neurotransmitter at the presynaptic active

Neurexin Cerebellin-1 GluRG2 N-Cadherin N-Cadherin

Presynaptic Syndecan F-Actin GRASP Homer Cortactin MARCKS mGlu R Shank SNAP-25 VAMP PSD-95 GKAP nNOS Neuritin Syntaxin-1 NMDA R Fyn Complexin + K Synaplophysin Synaptotagmin SALM4/5 vGlut Synapsin Dynamin SALM4/5 N-Acetyl-Asp-Glu LRRTM2 Neuroligin Ca2+ D-Synuclein Neurexin-1 Postsynaptic Glutamate Nectin-3 PSMA/FOLH1 Na+ Neurexin Nectin-1

Figure 24. Synaptic Proteins and Receptors Schematic. This illustration depicts key molecules for synaptic transmission. These include those related to presynaptic ion homeostasis, vesicle-mediated neurotransmitter release, and trans-synaptic cell adhesion. For more information regarding products for Neurotransmitters, Receptors, and Associated Enzymes, please see page 48.

44 For research use only. Not for use in diagnostic procedures. SYNAPTIC PROTEINS & NEUROTRANSMITTERS

R&D Systems Products for Synaptic Proteins RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Synaptic Proteins Fyn H (IHC, WB) M (WB) R (WB)

Agrin H R R (B/N, ELISA, WB) GRASP H (WB) M (WB) R (WB) B (WB) Ca (WB)

Amphiphysin/AMPH H (IHC, WB) M (WB) R (WB) HOMER1 H (WB) M (WB) R (WB)

BoNT-A Light Chain Ba Ba (IP, WB) Kynureninase HH (IP, WB)

BoNT-B Light Chain Ba Ba (IP, WB) Laminin S H (IHC, IP, WB) R (IHC, IP, WB) Ch (IHC, IP, WB)

BoNT-C1 Heavy Ba (IP, WB) MuSK H (WB) R (B/N, WB) Chain NCAM-1/CD56 H M H (ELISA, FC, IHC, WB) M (WB) R (WB) BoNT-D Heavy Chain Ba (WB) NCAM-L1/L1CAM H M H (IHC, WB) M (FC) BoNT-D Light Chain Ba (WB) Neurexophilin-1/ H R R (WB) BoNT-E Light Chain Ba Ba (WB) NXPH-1

BoNT-G Light Chain Ba (WB) Neurexophilin-3/ RH (WB) M (WB) R (WB) NXPH-3 BSRP-A H (WB) M (IHC, WB) Neuronal Pentraxin H (IHC, WB) BSRP-C M (IHC, WB) R/NPTXR

N-Cadherin H M H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) Neuroplastin H (IHC, WB)

Calsyntenin-1 H (WB) N-WASP H (IHC, WB)

Calsyntenin-2 H (IHC, WB) PSD-95 H (WB) M (WB) R (WB)

Calsyntenin-3 H (IHC, WB) SALM2/LRFN1 HH (IHC, WB) Caspr2 H (IHC, WB) SALM3/LRFN4 H

Cerebellin-1 H SALM4/LRFN3 HH (WB)

Cerebellin-2 H (IHC, WB) SHANK2 H (IHC)

Cerebellin-4 H (IHC) SNAP23 H (IHC, WB)

Clathrin Heavy Chain H (WB) SNAP25 H (WB) 1/CHC17 Synapsin I H (IF, IHC, IP, WB) M (IF, IHC, IP, WB) R (IF, IHC, IP, WB) Clathrin Heavy Chain H (WB) B (IF, IHC, IP, WB) Ca (WB) Ch (WB) X (WB) Z (WB) 2/CHC22 Synaptophysin H (IHC, WB) R (IHC, WB) Complexin-2 H (WB) M (WB) R (WB) Synaptotagmin-1 H (WB) M (WB) R (IHC, IP, WB) B (WB) Ca (WB) Ch (WB) DISC1 H (IHC, WB) Pr (WB) Z (WB)

DOK7 H (WB) M (WB) R (WB) Syndecan-1/CD138 H M H (ELISA, FC, IHC, WB) M (FC, IHC, IP, WB)

Dystroglycan H (IHC, WB) Syndecan-2 H M H (FC, IHC)

Epimorphin/ HH (WB) M (IHC, WB) Syntabulin H (WB) Syntaxin 2

Synaptophysin in Rat Medulla. Synaptophysin was detected in perfusion-fi xed BSRP-A in Mouse Hippocampus. Brain-specifi c receptor-like protein A (BSRP-A) Vanilloid R1/TRPV1 in Rat Dorsal Root Ganglion. Vanilloid R1/TRPV1 was frozen sections of rat brainstem using a Goat Anti-Human/Rat Synaptophysin was detected in perfusion-fi xed frozen sections of mouse brain using a Rat Anti- detected in perfusion fi xed frozen sections of rat dorsal root ganglia using a Goat Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5555). The tissue Mouse BSRP-A Monoclonal Antibody (Catalog # MAB4916). The tissue was stained Anti-Rat Vanilloid R1/TRPV1 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog was stained using the NorthernLights™ 557-conjugated Donkey Anti-Goat IgG with the Anti-Rat HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS017; brown) and # AF3066). The tissue was stained (red) and nuclei were counterstained (green). Secondary Antibody (Catalog # NL001; red) and nuclei were counterstained (green). nuclei were counterstained with hematoxylin (blue).

www.RnDSystems.com/go/Synapse 45 SYNAPTIC PROTEINS & NEUROTRANSMITTERS

R&D Systems Products for Synaptic Proteins RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Synaptic Proteins, continued VAMP-7 H (IHC, WB)

Syntaxin 5 H (WB) VAMP-8 H (WB)

Syntaxin 6 H (WB) VAP-A HH (WB)

Syntaxin 7 H (WB) M (WB) R (WB) VAP-B HH (IHC, WB)

Syntaxin 8 H (WB) M (WB) R (WB) VIAAT/SLC32A1 H (IHC, WB)

Syntaxin 12 H (WB) Contactins

Syntaxin 16 H (WB) Contactin-1 HH (B/N, IHC, WB)

Syntaxin 1B H (WB) M (WB) R (WB) Contactin-2/TAG1 H M H (IHC, WB) M (IHC, WB) R (IHC, WB)

Syntaxin-BP1 H (IHC, WB) M (IHC, WB) R (IHC, WB) Contactin-3 HH (IHC, WB) M (FC, WB) R (WB)

Syntaxin-BP2 H (WB) Contactin-4 H M H (B/N, WB) M (IHC, WB)

Syntaxin-BP3 H (WB) Contactin-5 HH (WB)

Synuclein-α H (IHC, WB) M (WB) R (WB) B (WB) Ca (WB) Pr (WB) Contactin-6 MM (IHC, WB)

Synuclein-β H (IHC, WB) IGSF4/SynCAM Family

Synuclein-γ H (IHC, WB) IGSF4A/SynCAM1 H M M (FC, WB)

Talin2 H (WB) IGSF4B/SynCAM3 HH (FC, IHC, WB)

Tau Ms (IHC, WB) IGSF4C/SynCAM4 HH (FC, IHC, WB)

α-Taxilin H (WB) IGSF4D/SynCAM2 H

TeNT Light Chain Ba (IP, WB) Nectins

Thrombospondin-2 HH (ELISA, WB) Nectin-1 HH (FC, WB)

VAMP-1 H (IHC, WB) M (IHC, WB) Nectin-2/CD112 H M H (FC, WB) M (WB)

VAMP-1/VAMP-2 H (IHC, WB) M (IHC, WB) Nectin-3 HH (FC, IHC, WB) M (WB)

VAMP-2 H (IHC, WB) M (IHC, WB) R (WB) Nectin-4 H M H (FC, IHC, WB) M (WB)

A. B.

Syntaxin-BP1 in Mouse Hippocampus. Syntaxin-binding protein 1 (Syntaxin-BP1) LRRTM2 in Embryonic Mouse Neural Tube. Leucine rich repeat transmembrane Calsyntenin-3 in Human Cortex. Calsyntenin-3 was detected in immersion- was detected in perfusion-fi xed frozen sections of mouse brain using a Goat Anti- protein 2 (LRRTM2) was detected in immersion-fi xed frozen sections of embryonic fi xed paraffi n-embedded sections of human cortex using a Sheep Anti-Human Human/Mouse/Rat Syntaxin-BP1 Antigen Affi nity-purifi ed Polyclonal Antibody mouse neural tube (E10.5) using a Sheep Anti-Human/Mouse LRRTM2 Antigen Calsyntenin-3 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5244). (Catalog # AF5675). The tissue was stained using the Anti-Goat HRP-DAB Cell & Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5589). The tissue was stained Before incubation with the primary antibody, the tissue was subjected to heat- Tissue Staining Kit (Catalog # CTS008; brown) and nuclei were counterstained with with the NorthernLights™ 557-conjugated Donkey Anti-Sheep IgG Secondary induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). hematoxylin (blue). Antibody (Catalog # NL010; red) and counterstained with DAPI (blue). The tissue was stained using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS019; brown) and nuclei were counterstained with hematoxylin (blue). Specifi c labeling was localized to the neuronal processes and synaptic boutons.

46 For research use only. Not for use in diagnostic procedures. SYNAPTIC PROTEINS & NEUROTRANSMITTERS

R&D Systems Products for Synaptic Proteins RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Neurexins & Neuroligins EN-RAGE/S100A12 HH (FC, IHC, WB)

Neurexin 1α/NRXN1a R Nesfatin-1/ H (IHC, WB) M (IHC, WB) R (IHC, WB) Nucleobindin-2 Neurexin 1β/NXRN1b HR (WB) Parvalbumin  H (IHC, WB) M (IHC, WB) R (IHC, WB) Neurexin 2α/NRXN2a H Polycystin-1/PKD1 H (IHC) Neurexin 3/NRXN3 H (IHC, WB) M (IHC, WB) S100A1 H (WB) Neurexin 3β/NRXN3b HH (IHC, WB) M (IHC, WB) S100A2 H (IHC, WB) Neuroligin 1/NLGN1 H R R (WB) S100A4 H M H (WB) M (IHC, WB) Neuroligin 2/NLGN2 HH (WB) R (WB) S100A6 H (WB) M (WB) Neuroligin 3/NLGN3 H (IHC, WB) M (IHC, WB) R (IHC, WB) S100A7 H (IHC, WB) Neuroligin 4/NLGN4 HH (IHC, WB) S100A8 H (FC, IHC, WB) M (IHC, WB) Calcium-binding Proteins & Related Molecules S100A9 H (IHC, WB) M (IHC, WB) Calbindin D H (IHC, WB) S100A10 H (IHC, WB) M (FC, IHC, WB) Calcitonin R H (FC) S100A11 H (IHC, WB) M (WB) Calreticulin H (FC, WB) S100A13 H M H (WB) M (IHC, WB) Calreticulin-2 H (FC, WB) S100A16 H (IHC, WB) M (WB) Calretinin H (IHC, WB) M (IHC, WB) R (IHC, WB) S100B H (IHC, WB) Calsyntenin-1 H (WB) S100P H (IHC, WB) Calsyntenin-2 H (IHC, WB) SMOC-1 H M M (WB) R (WB) Calsyntenin-3 H (IHC, WB) SMOC-2 H M H (IHC, WB) M (WB) CaM Kinase II H (WB) M (WB) R (WB) X (WB) Synaptotagmin-1 H (WB) M (WB) R (IHC, IP, WB) B (WB) Ca (WB) Ch (WB) CaM Kinase II H (IHC) Pr (WB) Z (WB)

CaM Kinase II H (IHC, WB) TPT1/TCTP H (WB) M (WB) R (WB) CaMKK H (WB) R (WB)

A. Synuclein- B. Syntaxin-BP1C. Phospho-Synapsin IA. NMDA NR2A B. GABA-A Receptor 3 C. Phospho-GluR1 an m u BM H C

. kDa ain an C at at ppo e R Br R S. m i ain kDa s u a

H 97 e kDa H Br 116 s at a ppo -PP i R kDa at R H O ain 95 PP kDa at O 39 kDa R Br

100 200 54 23 100 39 120 68 37 19 68 68 43 29 23 7 43 Detection of Presynaptic Markers. Western blots show lysates of the indicated tissue. A. The PVDF membrane was probed using Detection of Postsynaptic Markers. Western blots show lysates of the indicated tissue. A. The PVDF membrane was probed using a Sheep Anti-Human Synuclein- Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5745) followed by a HRP-conjugated a Rabbit Anti-Human/Mouse/Rat NMDA Receptor NR2A Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # PPS012) followed

Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). B. The PVDF membrane was probed using a Goat Anti-Human/Mouse/ by an anti-rabbit secondary antibody. B. The PVDF membrane was probed using a Sheep Anti-Human GABAA Receptor 3 Antigen Rat Syntaxin-BP1 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5675) followed by a HRP-conjugated Anti-Goat IgG Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5590) followed by a HRP-conjugated Anti-Sheep IgG Secondary Antibody Secondary Antibody (Catalog # HAF019). C. The PVDF membrane was probed using a Rabbit Anti-Human/Mouse/Rat Phospho- (Catalog # HAF016). C. The PVDF membrane was probed using a Rabbit Anti-Human/Mouse/Rat Phospho-GluR1 (S831) Antigen Synapsin I (S9) Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # PPS084) followed by an anti-rabbit secondary antibody. Affi nity-purifi ed Polyclonal Antibody (Catalog # PPS084) followed by an anti-rabbit secondary antibody. The phospho-specifi city The phospho-specifi city of this antibody was supported by decreased labeling following treatment with 1200 U -phosphatase of this antibody was supported by decreased labeling following treatment with 1200 U -phosphatase (-PPase) for 30 minutes. (-PPase) for 30 minutes. Abbreviation: spinal cord (S.C.). Abbreviations: hippocampus (Hippo), cerebellum (CBM).

www.RnDSystems.com/go/Synapse 47 SYNAPTIC PROTEINS & NEUROTRANSMITTERS

Neurotransmitters, Receptors, & Related Molecules Synaptic vesicles packed with neurotransmitter are released from the In contrast, group II (mGluR2 and 3) and group III (mGluR4, 6, 7, and 8) presynaptic active zone to facilitate the transmission of information to mGluRs are normally expressed on presynaptic terminals or preterminal the postsynaptic cell. Commonly studied neurotransmitter molecules axons where they function to inhibit neurotransmitter release.4 include acetylcholine, dopamine, -aminobutyric acid (GABA), glutamate,  The long-term stability and function of neuronal networks is dependent serotonin and nitric oxide. Although many neurotransmitters are on a maintained balance between excitatory and inhibitory synaptic considered inherently excitatory or inhibitory, their action in the target cell transmission.5 The major inhibitory neurotransmitter in the CNS is is dependent on postsynaptic receptor expression. Postsynaptic eff ects of GABA, which is converted from Glutamic Acid by the enzyme Glutamic neurotransmitters generally involve changes in membrane potential and/ Acid Decarboxylase (GAD). Similar to GluRs, GABA receptors are or activation of G protein-coupled signaling cascades. ionotropic (GABA-A) or metabotropic (GABA-B).6 GABA-A receptors are Glutamate receptors (GluRs), the major excitatory receptor in the brain, heteropentameric ligand-gated ion channels that selectively permit the – – are characterized as ionotropic or metabotropic. Ionotropic GluRs are infl ux of Cl and HCO3 ions to decrease membrane excitability. Extremely tetrameric ligand-gated cation channels that induce depolarization of the heterologous with at least nineteen known subunit genes, GABA-A postsynaptic membrane following the presynaptic release of glutamate receptors mediate the majority of fast synaptic inhibition. Metabotropic (Figure 25).1 Their actions underlie the cellular models of learning and GABA-B receptors are G protein-coupled heterodimers of GABA-B1 and memory, modulate the excitability of neuronal networks, and are required GABA-B2. They are expressed on both the presynaptic and postsynaptic for synaptic maturation.2 Ionotropic GluRs can be pharmacologically terminals where they inhibit neurotransmitter release and induce cell classifi ed according to their sensitivity to AMPA, Kainate, and NMDA. membrane hyperpolarization, respectively.7 AMPA receptors (GluR1–4) evoke excitatory postsynaptic potentials References and mediate fast (< 10 ms) synaptic transmission. In contrast, Kainate 1. Tomita, S. (2010) Physiology 25:41. receptors (GluR5-6 and KA1-2) and NMDA receptors (NR1-3) mediate 2. Swanson, G.T. & R. Sakai (2009) Prog. Mol. Subcell. Biol. 46:123. slower synaptic transmission (10–100 ms) and exert eff ects on plasticity. 3. Niswender, C.M. & P.J. Conn (2010) Ann. Rev. Pharmacol. Toxicol. 50:295. In addition, glutamate can modulate neuronal excitability and synaptic 4. Shigemoto, R. et al. (1997) J. Neurosci. 17:7503. transmission through second messenger signaling pathways. Meta- botropic glutamate receptors (mGluRs) are G protein-coupled receptors 5. Turrigiano, G. et al. (2007) Curr. Opin. Neurobiol. 17:318. that function as constitutive dimers.3 There are eight mGluR subtypes that 6. Filip, M. & M. Frankowska (2008) Pharmacol. Rep. 60:755. are diff erentially expressed in specifi c neuronal populations throughout 7. Schuler, V. et al. (2001) Neuron 31:47. the CNS. Metabotropic glutamate receptors are classifi ed into three 8. Eroglu, C. & B.A. Barres (2010) Nature 468:223. groups based on sequence homology, G protein-coupling, and ligand selectivity. In general, group I mGluRs (mGluR1 and 5) are expressed on postsynaptic membranes and function to increase neuronal excitability.

Plasma membrane Glial Cell GABA transporters Glutamine GAD Glutamate Glutamic Acid Glutaminase Glutamate Glutamine mGluR5 GABA Presynaptic vGlut Glutamate EAAT1 GABA Vesicular GABA transporter GAT mGluR3 mGluR4,6,7,8 Gi/o Gi/o 2 GABA-B 1b G G i/o NMDA AMPA i/o mGluR1/5 1a 2 mGluR2/3 Kainate GABA-A GABA-B G mGluR7 mGluR2/3 q Gi/o K+ G i/o Ca2+ u uNa+ uCl– ucAMP mGlu2/3 Ca2+ vcAMP Postsynaptic

Depolarization Hyperpolarization

Figure 25. Glutamate and GABA Synaptic Transmission. Glutamate evokes postsynaptic depolarization and excitation via ionotropic (AMPA, Kainate, and NMDA) and metabotropic (mGluR) glutamate receptors (left). In contrast, GABA induces postsynaptic hyperpolarization and inhibition via ionotropic (GABA-A) and metabotropic (GABA-B) receptors (right). Feedback from presynaptic receptors inhibits the excessive release of neurotransmitter. Extracellular glutamate and GABA molecules are also transported into glia (center), where they are converted to glutamine and recycled back to neurons.8 For more information about synaptic proteins and neurotransmitter release, please see page 44.

48 For research use only. Not for use in diagnostic procedures. SYNAPTIC PROTEINS & NEUROTRANSMITTERS

R&D Systems Products for Neurotransmitters, Receptors, & Related Molecules RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs Neurotransmitter Receptors GABARAP/Apg8p1 H

5HT1B H (IHC) GAD1/GAD67 H (IHC, WB)

5HT2C H (FC, IHC) GAD2/GAD65 H (IHC, WB)

5HT4R H (IHC) GATE-16/Apg8p2 H

AGTR-2 H (FC) Glutamate Receptors

BSRP-A H (WB) M (IHC, WB) GluR1 H (WB) M (WB) R (WB) Ca (WB) Ch (WB) Pr (WB)

BSRP-C M (IHC, WB) GluR2 H (WB) M (WB) R (WB) Ch (WB) Pr (WB) Z (WB)

Cannabinoid R1 H (FC) GluR2/3 H (WB) M (WB) R (WB) Ch (WB) Z (WB)

Cannabinoid R2/ H (FC, IHC) GluR3 H (WB) M (WB) R (WB) Ch (WB) Z (WB) CB2/CNR2 mGluR1a H (IHC, WB) M (IHC, WB) R (IHC, WB) CFTR H (ELISA, IF, IHC, IP, WB) mGluR2/3 H (WB) M (WB) R (WB) Ch (WB) Z (WB) CHRM2 H (IHC) mGluR2 H (FC, WB) M (WB) R (WB) Ch (WB) Z (WB) CHRM3 H (IHC) mGluR3 H (IHC, WB) M (WB) R (WB) Ch (WB) Z (WB) Galanin R2 H (IHC) mGluR5 H (FC, IHC, WB) M (IHC, WB) R (IHC, WB) Glycine R R (IHC, WB) mGluR5/1a H (IHC, WB) M (IHC, WB) R (IHC, WB) Growth Hormone R/ H M R H (B/N, FC, IHC, WB) M (IHC, WB) R (B/N, IHC, WB) GHR mGluR7 H (FC)

HCRTR2 H (FC, IHC) mGluR8 H (IHC, WB)

Histamine H1 R H (FC) NMDA R, NR1 H (IHC, IP, WB) M (IHC, IP, WB) R (IHC, IP, WB) Subunit KOR H (FC, IHC) NMDA R, NR2A H (IHC, IP, WB) M (IHC, IP, WB) R (IHC, IP, WB) Leptin R H M H (FC, IHC, WB) M (ELISA, FC, IHC, WB) H M Subunit

Leukotriene B4 R1 H (FC) NMDA R, NR2B H (IHC, IP, WB) M (IHC, IP, WB) R (IHC, IP, WB) Pr (WB) Subunit MRGX2 H (FC) NMDA R, NR2C H (IF, IHC, IP, WB) M (IF, IHC, IP, WB) R (IF, IHC, IP, WB) NPY1R H (FC) Subunit OPRM1 R (WB) Neurotransmitter Transporters & Ion Channels

PAR1 H (FC, IHC, WB) Dopamine H (IHC, WB) M (IHC, WB) Pr (IHC, WB) Transporter PAR2 H (FC) DPP6 H (FC, IHC, IP, WB) SEZ6L H (WB) M (IHC, WB) DPP10 H (WB) Vanilloid R1/TRPV1 R (IHC) EAAT1/GLAST-1 H (IHC, IP, WB) Vanilloid R-like 3/ H (IHC) TRPV3 Glut1 H (FC, IHC)

VIP R2 H (IHC) Glut2 H (FC, IHC) M (FC, IHC)

GABA Receptors Glut3 H (FC, IHC)

FAT10 H Glut4 R (AP, IHC, IP, WB) (WB) (WB) (WB) (WB) (WB) (WB) (WB) GABAA R 1 H M R B Ca Pr Z Glut5 H (FC, IHC) (WB) (WB) (WB) (WB) (WB) (WB) GABAA R 2 H M R B Ca Pr Nucleoporin NUP85 H (IHC, WB) (WB) (WB) (WB) (WB) (WB) (WB) (WB) GABAA R 4 H M R B Ca Pr Z Polycystin-1/PKD1 H (IHC) (WB) (WB) GABAA R 5 M R Potassium Channel R (IHC, WB) X (IHC, WB) Kv2.2 (WB) (WB) (WB) (WB) GABAA R 6 H M R B Potassium Channel M (IHC, WB) R (IHC, WB) GABA R 1 H (WB) M (WB) R (WB) A Kv3.1 GABA R 2 H (WB) M (WB) R (WB) B (WB) Ca (WB) Pr (WB) A SLC22A1 H (WB) GABA R 3 H (WB) M (WB) R (WB) A SLC22A2 H (IHC) GABA R 2 H (WB) M (WB) R (WB) B (WB) Ca (WB) Ch (WB) A Sodium Calcium Ms (WB) Pr (WB) Z (WB) Exchanger 1/NCX1 GABA R  M (IHC, IP, WB) R (IHC, IP, WB) A VIAAT/SLC32A1 H (IHC, WB) (IF, WB) (IF, WB) (IF, IHC, WB) (IF, WB) (IF, WB) GABAB R2 H M R B Ca Ch (IF, WB) Pr (IF, WB) X (IF, WB) Z (IF, WB)

Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: AP Affi nity Purifi cation B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot

www.RnDSystems.com/go/Neurotransmitters 49 NEURODEGENERATIVE DISEASES

Alzheimer’s Disease & APP Metabolism Alzheimer’s disease (AD) aff ects approximately 10% of the population which increase amyloidogenic processing of APP, cause the majority of over the age of sixty-fi ve, making it the most prevalent neurodegenerative early-onset familial AD (FAD).4 disorder.1 This devastating condition is characterized by progressive In addition to A generation, recent studies suggest that PS-1 aff ects the cog nitive impairment and neurodegeneration, commencing in the clearance of A. Farfara et al. discovered that interfering with PS-1 function hippo campus and cortex. Neuropathological hallmarks of AD include impaired the phagocytosis of soluble A by mouse microglia.5 In parallel, neuronal loss in the presence of neurofi brillary tangles and senile Jayadev et al. reported that knockdown of PS-2 in mouse microglia plaques. Neurofi brillary tangles are intraneuronal inclusions composed decreased -Secretase activity.6 This study also found that impairment of insoluble hyper phosphorylated forms of the microtubule protein Tau. of PS-2 function resulted in an exaggerated microglial release of TNF- Senile plaques represent extracellular deposits of aggregated Amyloid-  and IL-6, suggesting PS-2 dysfunction may also contribute to neuro- protein (A ), the proposed causative agent of AD. Prior to its deposition as  infl ammation during AD. Further reports suggest a novel role for PS-1 in senile plaques, A oligomerizes to exert pathological actions on neuron  axon guidance. PS-1 was shown to be required for the processing of DCC function and viability.2 receptors, which are essential for Netrin-mediated chemoattraction.7 A is generated by sequential enzymatic processing of Amyloid Precursor  AD therapies that are in the advanced stages of development mostly Protein (APP), a type I transmembrane protein. The majority of APP target A production, aggregation, or clearance. Although most clinical is cleaved by -Secretase, which does not produce A and is termed   trials focus on the treatment of symptomatic patients, it has been non-amyloidogenic (Figure 26). Putative -Secretases include ADAM9,  suggested that earlier intervention or disease prevention would be a ADAM10, and ADAM17/TACE.1 In contrast, a small percentage of APP more eff ective strategy.8 Research studies have investigated enzymes is processed by -Secretases, such as BACE-1 and Cathepsin D, which  that are known to degrade A (i.e. ACE, Cystatin C, ECE, IDE, MMPs, yields soluble APP (sAPP ) and a -C-terminal Fragment ( -CTF). The     Neprilysin, Plasmin) and receptors that are involved in the uptake of A -CTF fragment is further cleaved by -Secretase to generate A . The    (i.e. APP, CD36, FPRL, LDL R, LRP1,  -Macroglobulin, MARCO, SR-A1, and activity of -Secretase requires four components; Presenilin (PS, PS-1 or 2  RAGE). In fact, a recent report suggested that a combinatorial therapy PS-2), Presenilin Enhancer-2 (PEN-2), Nicastrin, and Anterior Pharynx- designed to block A production and enhance A clearance might defective-1 (Aph-1). Primarily located in the endoplasmic reticulum, represent the most eff ective approach.9 trans-Golgi network and endosomes, -Secretase activity is modulated by additional factors, including CD147/EMMPRIN.3 Mutations in APP and PS, Recent mechanistic studies revealed novel targets for AD prevention and treatment. In a transgenic mouse model of AD (J20), oligomers of A disrupted NMDA currents by binding and triggering the degradation of INTERNALIZATION PROTEASOMAL DEGRADATION EphB2 receptor tyrosine kinase.10 In contrast, Complement Protein C1q EphB2 was recently shown to protect against A-induced neurodegeneration via the upregulation of NGF and NT-3.11 Interestingly, knockout of NGF NMDA R Postsynaptic receptor p75 (NGF R) reduced levels of soluble A and increased its deposition as senile plaques, an eff ect that was independent of changes in secretase activity.12 In another report, small molecule enhancer of AE Degradation: ACE, Cystatin C, ECE, IDE, rapamycin 28 (SMER28) stimulated autophagy-dependent degradation of Plasmin, MMPs, Neprilysin A, via Atg5, Beclin1, and Ulk1.13 OLIGOMERIZATION References APP AE Uptake: 1. Zhang, Y. et al. (2011) Mol. Brain 4:3. CD36, LDL R, FPRL, LRP1, D2-Macroglobulin, sAPPD MARCO, SR-A1, RAGE, APP 2. Sakono, M. & T. Zako (2010) FEBS J. 277:1348. AE 3. Zhou, S. et al. (2005) Proc. Natl. Acad. Sci. USA 102:7499. Presynaptic TACE 4. Bertram, L. et al. (2010) Neuron 68:270. D-CTF 5. Farfara, D. et al. (2011) Ann. Neurol. 69:170.

6. Jayadev, S. et al. (2010) PLoS One 5:e15743. -Secretase BACE J 7. Bai, G. et al. (2011) Cell 144:106. p3 APP AE 8. Golde, T.E. et al. (2011) Neuron 69:203. AICD AICD 9. Wang, A. et al. (2011) J. Neurosci. :4124. Endosome BACE 31 10. Cissé, M. et al. (2011) Nature 469:47. sAPPE Trans-Golgi Network E-CTF 11. Benoit, M.E. & A.J. Tenner (2011) J. Neurosci. 31:3459.

12. Wang, Y.J. et al. (2011) J. Neurosci. 31:2292.

Figure 26. APP Metabolism & A Clearance. Under normal physiological conditions, the majority of APP undergoes non- 13. Tian, Y. et al. (2011) FASEB J. 25:1934. amyloidogenic cleavage by -Secretase (TACE) (blue arrows). In parallel, a small proportion of APP is processed by -Secretase (BACE) and -Secretases to generate A, which oligomerizes and exerts detrimental eff ects on neuron function and viability (green arrows). Recent studies suggest that oligomeric A may impair synaptic function by binding to EphB2, which leads to EphB2 degradation and NMDA receptor internalization.10

50 For research use only. Not for use in diagnostic procedures. NEURODEGENERATIVE DISEASES

R&D Systems Products for Alzheimer’s Disease & APP Metabolism RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Acetylcholinesterase/ M ECE-1 H M H (FC, IHC, IP, WB) ACHE ECE-2 HH (IHC, IP, WB) ADAM9 H M H (ELISA, FC, IP, WB) M (FC, IHC, IP, WB) H FPRL1 H (FC) ADAM10 H M H (FC, IHC, IP, WB) M (FC, IHC, WB) F-Spondin/SPON1 HH (B/N, WB) ADAMTS4 HH (B/N, IP, WB) Glutaminyl-peptide H APBA2 H (WB) M (WB) R (WB) Cyclotransferase/QPCT

APBA3 H (WB) Insulysin/IDE H R H (FC, IP, WB)

APH1A H (IHC) ITM2B H (IHC)

APLP-1 H (FC, IHC, IP, WB) M (IP, WB) ITM2C H (WB)

APLP-2 H (WB) M (WB) Kallikrein 6/Neurosin HH (IHC, IP, WB)

Apolipoprotein E/ApoE H (WB) LRRTM3 H M H (IHC, WB) M (IHC, WB)

APP/Protease Nexin II HH (IHC, IP, WB) M (IHC, WB) R (WB) Ca (WB) H M R MMP-2 H M R H (FC, IHC, IP, WB) M (IHC, IP, WB) R (IHC, IP, WB) H Ch (WB) Pr (WB) Ms (WB) MMP-9 H M R H (ELISA, FC, IHC, IP, WB) M (ELISA, IHC, IP, WB) H M APP 695+1 H (IHC, IP, WB) Neprilysin/CD10 H M H (ELISA, FC, IHC, IP, WB) M (B/N, ELISA, IHC, IP, WB) H M APP+1 HH (WB) Neurolysin HH (IP, WB) M (IP, WB) R (IP, WB) Arylsulfatase A/ARSA H M H (IHC, IP, WB) Nicastrin H (IHC, WB) BACE-1 H M H (B/N, FC, IHC, IP, WB) M (B/N, FC, IHC, IP, WB) Plasminogen HH (B/N, IP, WB) BACE-2 MH (IHC, IP, WB) M (IP, WB) Presenilin-1 H (IHC, WB) H Cathepsin B H M H (ELISA, IHC, IP, WB) M (B/N, IHC, WB) H Presenilin-2 H (IHC, WB) Cathepsin D H M H (IHC, IP, WB) M (IHC, IP, WB) PSENEN Ms (IHC) Cathepsin E H M H (IHC, IP, WB) M (IHC, IP, WB) RAGE H M R Ca H (B/N, ELISA, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M R Ca Clusterin H M H (ELISA, IHC, IP, WB) M (ELISA, IHC, IP, WB) H M R (B/N, ELISA, IHC, WB) Ca (ELISA, WB)

Complement H (IHC, WB) Serpin A3/ HH (IP, WB) Component C1qC α1-Antichymotrypsin

Cystatin C H M R H (B/N, ELISA, IHC, IP, WB) M (ELISA, IHC, IP, WB) H M Serpin A3N MM (IP, WB) R (IHC, IP, WB) Serpin E2/PN1 H M H (WB) M (B/N, IP, WB) DISC1 H (IHC, WB) TACE/ADAM17 H M H (ELISA, FC, IHC, IP, WB) H DYRK1A H (IHC, WB) R (IHC, WB) Thimet Oligopeptidase/ HH (IHC, IP, WB) DYRK2 H (IHC, WB) M (WB) R (IHC, WB) THOP1

7 d zol a eate r nt 6 U Nocod Phospho-APP 5 APP 4

2 Phospho-APP (ng/mg Lysate)

0 Untreated Nocodazole

Measurement of Phospho-APP (T668) levels using the DuoSet® IC ELISA. Lysates RAGE in Mouse Spinal Cord. Receptor for Advanced Glycation End Products (RAGE) Clusterin in Alzheimer’s Disease Brain. Clusterin was detected in immersion-fi xed prepared from rat C6 cells that were either untreated or treated with nocodazole, was detected in perfusion-fi xed frozen sections of mouse spinal cord using a Rat paraffi n-embedded sections of human Alzheimer’s disease brain using a Goat Anti- were quantifi ed using the Human/Mouse/Rat Phospho-APP (T668) DuoSet IC ELISA Anti-Mouse RAGE Monoclonal Antibody (Catalog # MAB11794). The tissue was Human Clusterin Isoform 1 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # (Catalog # DYC2508; bar graph). The same lysates were also immunoblotted (inset) stained using the NorthernLights™ 557-conjugated Goat Anti-Rat IgG Secondary AF2937). The tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining using either a Rabbit Anti-Human/Mouse Phospho-APP (T668) Affi nity Purifi ed Antibody (Catalog # NL013; red) and counterstained with DAPI (blue). Specifi c Kit (Catalog # CTS008; brown) and counterstained with hematoxylin (blue). Polyclonal Antibody (Catalog # AF2508) or a Goat Anti-Human APP Pan Specifi c staining was localized to the plasma membrane in motor neurons. Affi nity Purifi ed Polyclonal Antibody. The DuoSet IC ELISA results correlate well with the relative amounts of phosphorylated APP detected by Western blot.

www.RnDSystems.com/go/AD 51 NEURODEGENERATIVE DISEASES

Parkinson’s Disease Parkinson’s disease (PD) is a progressive neurodegenerative condition that Mitochondrial dysfunction is thought to be central to the pathogenesis of is characterized by the death of dopaminergic neurons projecting from PD. This theory is supported by the fact that mutations in mitochondrial the substantia nigra pars compacta (SNc) to the striatum. In addition to PTEN-induced Kinase 1 (PINK1), Parkin, HTRA2/Omi, and Park7/DJ-1 result neuron loss, the major neuropathological hallmark of PD is the deposition in recessive forms of the disease. Recent studies suggest that PINK1 is of Lewy bodies throughout the nigrostriatal pathway and in other parts responsible for the disposal of dysfunctional mitochondria, which leak of the brain.1 Lewy bodies are intracellular inclusions composed of harmful ROS.12 Removal of defective mitochondria by macroautophagy -Synuclein that appear in an ascending course from the brain stem to requires relocation of Parkin from the cytoplasm to the mitochondria.12 the cortex. Although aging is the greatest risk factor for the development However, overactivation of Parkin-dependent mitochondrial autophagy of PD, early-onset familial forms, caused by mutations in twelve known (mitophagy) may lead to a bioenergetic defi cit that contributes to loci, represent approximately 10% of all PD cases. Autosomal dominant neurodegeneration.13,14 mutations in other genes, such as LRRK2, also increase the risk of Current treatments for PD include L-DOPA and dopamine agonists, developing sporadic late onset PD.2 which are very eff ective at reversing motor symptoms but do not How -Synuclein aff ects dopamine homeostasis in the SNc has been an prevent the progression of the underlying neurodegeneration. A novel area of intense study (Figure 27).3 It is known that -Synuclein inhibits strategy to couple symptom relief with neuroprotection is the use of tyrosine hydroxylase (TH), the rate limiting enzyme that converts tyrosine group III metabotropic glutamate receptor (mGluR) agonists. Activation to L-3,4-dihydroxyphenylalanine (L-DOPA).4 The presence of -Synuclein of presynaptic group III mGluRs inhibits excessive glutamate release, also prevents the conversion of L-DOPA to dopamine by inhibiting Dopa prevents excitotoxicity, and combats motor symptoms.15 Decarboxylase (DDC).5 In addition to reducing dopamine synthesis, References

-Synuclein has been shown to impair dopaminergic vesicle traffi cking 1. Braak, H. et al. (2004) Cell Tissue Res. 318:121. and synaptic function by disturbing vesicle fusion and neurotransmitter 2. Greggio, E. et al. (2011) Mol. Neurodegener. 6:6. release.6 The availability of the ready releasable pool of dopaminergic vesicles is also lessened due to the eff ects of -Synuclein on the rapid 3. Venda, L.L. et al. (2010) Trends. Neurosci. 33:559. shut tling of the Dopamine Transporter (DAT) to and from the presynaptic 4. Perez, R.G. et al. (2002) J. Neurosci. 22:3090. 7 plasma membrane. 5. Tehranian, R. et al. (2006) J. Neurochem. 99:1184. Research has attempted to determine why some neuronal populations 6. Cabin, D.E. et al. (2002) J. Neurosci. 22:8797. are vulnerable to neurodegeneration during PD while others appear to 7. Wersinger, C. & A. Sudhu (2003) Neurosci. Lett. 340:189. be protected. It has been suggested that the long axons (up to 70 cm), 8. Matsuda, W. et al. (2009) J. Neurosci. 29:444. numerous synaptic connections, and high metabolic demands make mesostriatal dopaminergic neurons especially vulnerable.8 Alternatively, 9. Damier, P. et al. (1999) Brain 122:1421. lack of Calbindin expression may underlie the inability of A9 nigro- 10. Richardson, J.R. et al. (2006) FASEB J. 20:1695. striatal neurons to buff er calcium levels and increases susceptibility to 11. Fountain, T.M. et al. (2008) Eur. J. Neurosci. 28:2459. 9 calcium dependent neurotoxicity. Because dopamine rapidly undergoes 12. Narenda, D.P. et al. (2010) PLoS Biol. 8:e10002989. oxidation to form reactive oxygen species (ROS), cytoplasmic Dopamine 13. Choubey, V. et al. (2011) J. Biol. Chem. 286:10814. is rapidly sequestered into vesicles by Vesicle Monoamine Transporter 2 (VMAT2). Thus, neurons with a high DAT:VMAT2 ratio are thought to 14. Youle, R.J. & D.P. Narenda (2011) Nat. Rev. Mol. Cell. Biol. 12:9. be more vulnerable to neurodegeneration.10 Not only is the activity of 15. Duty, S. (2010) Brit. J. Pharmacol. 161:271. VMAT2 aff ected by -Synuclein, but small oligomeric accumulations of -Synuclein coalesce as insoluble cytotoxic protofi brils that perforate vesicular membranes and promote Dopamine release.11

n Tyrosine p Regulated by changes in: TH 1. Transcription D-Synuclein 2. Alternative Splicing L-DOPA 3. Micro RNA binding DDC q mGluRIII Dopamine q o r HTRA2 Park7 -Synuclein D-Synuclein Ub Calbindin Dopamine R D n Lewy body D-Synuclein G-protein o D-Synuclein PINK1 Nucleus Ub D-Synuclein Parkin Tau r p D-Synuclein Ca2+ D-Synuclein VMAT2 Mitochondria NF DAT

Figure 27. -Synuclein & Dopamine Homeostasis. A summary of the key cellular events during Parkinson’s disease including the upregulation of -synuclein , Lewy body formation , Dopamine synthesis , Dopamine transport and vesicle traffi cking , and mitochondrial dysfunction . Abbreviations: Ubiquitin (Ub), Neurofi lament (NF).

52 For research use only. Not for use in diagnostic procedures. NEURODEGENERATIVE DISEASES

R&D Systems Products for Parkinson’s Disease RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Calbindin D H (IHC, WB) TOR H (FC, IHC, IP, WB) M (FC, IHC, IP, WB) R (IP, WB) H

CDNF H M H (IHC, WB) M (IHC, WB) Tyrosine Hydroxylase M (IF, IHC, WB) R (IF, IHC, WB) Ms (IF, IHC, IP, WB)

DARPP-32 H (WB) M (IHC, WB) R (IHC, WB) UBE2E3 H

Dopa Decarboxylase/ HH (IHC, IP, WB) R (WB) B (WB) Ca (WB) UBE2K/E2-25K HH (IHC, WB) M (IHC, WB) R (WB) DDC Rb (WB) UBE2N (Ubc13)/ H Dopamine H (IHC, WB) M (IHC, WB) Pr (IHC, WB) Uev1a Complex Transporter UBE2N/Ubc13 H Dopamine H (WB) M (WB) Pr (WB) β-Hydroxylase Ubiquilin 1 H

GDNF H R H (B/N, ELISA, IHC, WB) R (B/N, IHC, WB) H Ubiquitin H Pz Pl Y Ms H (IHC, WB)

HTRA2/Omi HH (IHC, WB) M (IHC, WB) R (IHC, WB) Ubiquitin/ H (IHC, WB) Ubiquitin+1 LRRK2 H (IHC) Ubiquitin+1 HH (ELISA, IHC, WB) H NF-HH (IHC, WB) Di-Ubiquitin/Ub2 H Ms NF-L H (IHC, WB) Tri-Ubiquitin/Ub3 H NF-M H (IHC, WB) Tetra-Ubiquitin/Ub4 H N-Me-6,7-diOH-TIQ Ms (IHC) Penta-Ubiquitin/Ub5 H p53 HH (ChIP, FC, IHC, IP, WB) M (ChIP, IP, WB) R (ChIP, IP, WB) H M Hexa-Ubiquitin/Ub6 H Park7/DJ-1 H (IHC, WB) M (WB) H Octa-Ubiquitin/Ub8 H Parkin HH (IHC, WB) Poly-Ubiquitin H PINK1 H (IHC) UCH-L1 H M H (IHC, IP, WB) M (WB) R (WB) Synuclein- H (IHC, WB) M (WB) R (WB) B (WB) Ca (WB) Pr (WB) UCH-L3 H Rb H (WB) M (WB) R (WB) Synuclein- H (IHC, WB)

Synuclein- H (IHC, WB)

39 DARPP-32

Dopaminergic Neuron Diﬀ erentiation. Dopaminergic neurons generated using Detection of Mouse DARPP-32 by Western Blot. Dopamine- and cAMP-Regulated Synuclein- in Human Globus Pallidus. Synuclein- was detected in immersion- the Dopaminergic Neuron Diff erentiation Kit (Catalog # SC001B) were immersion- Phosphoprotein, Mr 32 kDa (DARPP-32) was detected in lysates of mouse brain fi xed paraffi n-embedded sections of human brain using a Sheep Anti-Human fi xed and incubated with a mouse anti-tyrosine hydroxylase monoclonal antibody tissue (corpus striatum) by Western blot. The PVDF membrane was probed using a Synuclein- Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF5528). The followed by the NorthernLights™ 493-conjugated Donkey Anti-Mouse IgG Rat Anti-Mouse/Rat DARPP-32 Monoclonal Antibody (Catalog # MAB4230) followed tissue was stained using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (Catalog Secondary Antibody (Catalog # NL009; green). In addition, cells were incubated by a HRP-conjugated Goat Anti-Rat IgG Secondary Antibody (Catalog # HAF005). # CTS019; brown) and counterstained with hematoxylin (blue). Specifi c labeling was with a NorthernLights 577-conjugated Mouse Anti-Neuron-specifi c -III Tubulin DARPP-32 was detected at approximately 32 kDa (as indicated). localized to the presynaptic terminals. Monoclonal Antibody (Clone TuJ-1) (Catalog # NL1195R; red). The nuclei were counterstained with DAPI (blue).

www.RnDSystems.com/go/PD 53 NEURODEGENERATIVE DISEASES

Amyotrophic Lateral Sclerosis Amyotrophic Lateral Sclerosis (ALS) is a fatal and incurable neurological ALS is an area of intense study. Overexpression of TDP-43 in the NSC34 disorder characterized by the selective degeneration of motor neurons in mouse neuroblastoma cell line was shown to induce cell death that the spinal cord, brainstem, and motor cortex. This devastating adult-onset was associated with an increase in pro-apoptotic BIM and a decrease in disease induces progressive weakness, muscle wasting, and death within anti-apoptotic Bcl-xL (Figure 28, part 2).8 Further studies suggested that three to fi ve years of diagnosis. The most prominent histopathological Ataxin-2 may enhance TDP-43 neurotoxicity to drive the motor neuron feature of ALS is the deposition of ubiquitinated TAR DNA-binding protein loss observed during ALS.9 43 (TDP-43) as intracellular inclusions.1 Although the majority of ALS Because ALS therapies increase life expectancy by just a few months, cases are sporadic, approximately 10-20% of cases result from familial researchers are investigating novel therapeutic strategies, including the mutations. The most common of these genes is copper-zinc superoxide delivery of neurotrophic factors in SOD1G93A mice. For example, Dodge dismutase (SOD1).2 In addition, recent studies discovered disease-causing et al. reported that virus-mediated expression of IGF-1 or VEGF delayed mutations in the genes for TDP-43 and FUS/TLS.3,4 motor decline and extended survival.10 In parallel, viral delivery of G-CSF Transgenic mice expressing mutant human SOD1, in particular the G93A to spinal cord neurons also delayed disease progression and increased mutation (SOD1G93A), have provided a critical tool for understanding the lifespan in this model.11 Additional studies reported that administration pathogenesis of ALS. SOD1 is a ubiquitously expressed antioxidant that of a recombinant IL-1 receptor antagonist improved motor function and catalyzes the disproportionation of superoxide radicals to hydrogen delayed the death of SOD1G93A mice.12 Finally, Ryu et al. proposed GDNF peroxide and molecular oxygen (Figure 28, part 1). Current research as a candidate therapeutic agent following their fi nding that oxidative suggests that mutations in SOD1 cause conformational changes that stress reduces c-Ret expression and impairs trophic GDNF signaling in induce mitochondrial dysfunction and disruption of axonal transport. vulnerable motor neuron populations.13 Disturbed mitochondrial function and axonal transport are consistent References with the dying-back axonopathy model of ALS.5 The dying-back hypothe- 1. Neumann, M. et al. (2006) Science 314:130. sis states that the denervation of motor neurons from muscle is an 2. Rosen, D.R. et al. (1993) Nature 362:59. early pathological event during ALS. Transgenic mice that specifi cally 3. Sreedharan, J. et al. (2008) Science 319:1668. overexpress Uncoupling Protein 1 (UCP1) in muscle tissue display an 4. Kwiatowski, T.J. et al. (2009) Science 323:1205. age-dependent deterioration of the neuromuscular junction (NMJ).6 Denervation of muscle tissue is followed by motor neuron pathology, 5. Dudon-Nachum, M. et al. (2011) J. Mol. Neurosci. 43:470. suggesting that distal events at the NMJ can induce subsequent motor 6. Dupuis, L. et al. (2009) PLoS One 4:e5390. neuron degeneration. 7. Bosco, D.A. et al. (2010) Nat. Neurosci. 13:1396.

Latest research suggests that the oxidation of wild type SOD1 causes 8. Suzuki, H. et al. (2011) J. Biol. Chem. 286:13171. a conformational change that is shared with ALS-linked mutant 9. Elden, A.C. et al. (2010) Nature 466:1052. forms of SOD1.7 Using vesicle motility assays in squid axoplasm, this 10. Dodge, J.C. et al. (2010) Mol. Therapy 18:2075. study found that oxidized SOD1 and mutant SOD1G93A both inhibited anterograde fast axonal transport, presenting a shared link between 11. Henriques, A. et al. (2011) Mol. Therapy 19:284. familial and sporadic ALS (Figure 28, part 4). Importantly, these defi cits 12. Meissner, F. et al. (2010) Proc. Natl. Acad. Sci. USA 107:13046. were observed in presymptomatic mice, suggesting axonal transport 13. Ryu, H. et al. (2011) Lab. Invest. 91:342. may be a key pathological event and early indicator of motor neuron degeneration. The cellular mechanism underlying neuronal death during

o Anterograde Glial Cell Ca2+ homeostasis APOPTOSIS q

rBcl-xL SOD1 Muscle Tissue qBIM Cytochrome c Dynactin Dynein r Kinesin Mitochondria p SOD1 p r EAAT1 n Lewy body o Retrograde – – + 02 + 02 + 2H TDP-43 FUS n Tau q SOD1 SOD1 Ub + Optineurin TDP-43 D-Synuclein SOD-1 H202 + 02 Ataxin-2 SOD1 Nucleus Glutamate transporter Ca2+

Figure 28. SOD1 Dysfunction & Motor Neuron Degeneration. A summary of key events during ALS pathogenesis including SOD1 activity , mitochondrial dysfunction , TDP-43 mislocalization and aggregation , axonal transport impairment , and muscular denervation . Abbreviations: Ubiquitin (Ub), Phosphorylation site (P).

54 For research use only. Not for use in diagnostic procedures. NEURODEGENERATIVE DISEASES

R&D Systems Products for Amyotrophic Lateral Sclerosis RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Aldehyde Oxidase 1/ H (WB) Ret H M H (FC, IHC, WB) M (IHC, WB) H AOX1 SOD (Superoxide Ms Ataxin UIM Domains H Dismutase)

Ataxin-3 H SOD1/Cu-Zn SOD H (IHC, WB) M (WB) R (WB)

Cytochrome c HH (IHC, IP, WB) M (IP, WB) R (IP, WB) E (IHC, IP) H M R SOD2/Mn-SOD H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R

Dynactin Subunit 1/ H (IHC, WB) Synuclein- H (IHC, WB) M (WB) R (WB) B (WB) Ca (WB) Pr (WB) DCTN1 Synuclein- H (IHC, WB) Dynactin Subunit 2/ H (IHC, WB) M (IHC, WB) DCTN2 Synuclein- H (IHC, WB)

EAAT1/GLAST-1 H (IHC, IP, WB) UBE2K/E2-25K HH (IHC, WB) M (IHC, WB) R (WB)

G-CSF H M H (B/N, ELISA, FC, WB) M (B/N, ELISA, WB) H M UCP1 H (WB) M (WB)

GDNF H R H (B/N, ELISA, IHC, WB) R (B/N, IHC, WB) H VAP-B HH (IHC, WB)

HSP70/HSPA1A H (IHC, WB) M (IHC, WB) R (WB) H M R VEGF H M R Ca F Z H (B/N, ELISA, FC, IF, IHC, WB) M (B/N, ELISA, IHC, WB) H M R Ca R (B/N, ELISA, IHC, WB) Ca (B/N, ELISA, IHC, WB) Z (B/N, WB) IGF-I H M R H (B/N, ELISA, IHC, WB) M (B/N, ELISA, IHC, WB) H M R

p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M

A.T B. 3 3 70

kDa NIH-

kDa NIH- 3 65 60 65 50 39 2 tBID + Bcl-x L 39 40 Total Untreated 30 tBID 23

1 Number Cell Relative 20 24 SOD2 Cytochrome c Released (O.D) Cytochrome SOD1 19 10 19 0 0 00.11 10 100 1,000 100 101 102 103 104

Concentration of Bcl-xL (nM) Ret

Measurement of Cytochrome c Levels using the DuoSet®IC ELISA. Isolated Detection of Mouse SOD1 and SOD2 by Western Blot. Western blots show lysates Detection of Ret by Flow Cytometry. The SH-SY5Y human neuroblastoma cell mouse liver mitochondria were treated with 60 nM Recombinant Human BID of the NIH-3T3 mouse embryonic fi broblast cell line. A. The PVDF membrane was line was stained with a Mouse Anti-Human Ret Monoclonal Antibody (Catalog

(Catalog # 882-B8) to induce Cytochrome c release. Cytochrome c release induced by probed with a Goat Anti-Human/Mouse SOD1/Cu-Zn SOD Antigen Affi nity-purifi ed # MAB718, fi lled histogram) or a Mouse IgG1 Isotype Control (Catalog # MAB002, BID is inhibited by increasing concentrations of Recombinant Mouse Bcl-xL (Catalog Polyclonal Antibody (Catalog # AF3418) followed by a HRP-conjugated Donkey open histogram), followed by an AP-conjugated Goat Anti-Mouse IgG Secondary # 878-BC). The amount of Cytochrome c released was measured using the Mouse/ Anti-Goat IgG Secondary Antibody (Catalog # HAF109). B. The PVDF membrane Antibody (Catalog # F0101B). To facilitate intracellular staining, cells were fi xed with Rat Total Cytochrome c DuoSet IC ELISA (Catalog # DYC897). was probed with a Goat Anti-Human/Mouse/Rat SOD2/Mn-SOD Antigen Affi nity- paraformaldehyde and permeabilized with saponin. purifi ed Polyclonal Antibody (Catalog # AF3419) followed by a HRP-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # HAF109). SOD1 and SOD2 were detected at approximately 19 kDa and 23 kDa, respectively (as indicated).

www.RnDSystems.com/go/ALS 55 NEURODEGENERATIVE DISEASES

Multiple Sclerosis Multiple sclerosis (MS) is a neurodegenerative autoimmune disorder degeneration through the release of infl ammatory cytokines (Figure 29). that is characterized by demyelination of neurons in the brain and spinal Tissue damage reveals novel antigens to DCs that induce a new wave of cord.1 The leading cause of disability in young adults, disease onset occurs autoimmune attack. This vicious cycle is termed ‘epitope spreading’ and suddenly between twenty and forty years of age. Relapsing-remitting MS underlies the relapsing phases of RRMS.9 (RRMS), the most common form of the disease, represents approximately There is no cure for MS. Current therapeutic options are restricted to 85% of MS patients. During RRMS, patients experience periods of disease modifying agents that off er symptomatic management only. exacerbation that are followed by a partial or complete recovery of Established treatment options, such as IFN-, signifi cantly improve MS defi cits. RRMS may evolve into secondary progressive MS (SPMS) and progression by modulating lymphocyte proliferation and directing the primary progressive MS (PPMS), which aff ect approximately 30% and 10% T cell response toward immunosuppressive Th2 and Treg cells.10 of patients with MS, respectively. Although patients with SPMS initially Fingolimod, the fi rst orally administered MS drug, was approved by the experience periods of remittance, both conditions are defi ned by a United States Food and Drug Administration in 2010. Fingolimod is a progressive decline, in the absence of recovery phases. The development Sphing osine-1 Phosphate Receptor agonist that attenuates immune of SPMS and PPMS indicates that extensive neuronal loss and axonal reactions by sequestering lymphocytes in the lymph nodes.11 Additional degeneration has occurred. studies are investigating mechanisms to induce the remyelination of The cause of MS is unknown. The current hypothesis states that auto- damaged neurons to restore neurological function.12 Failure to remyelin- immunity to CNS antigens is triggered by environmental factors in ate during MS may be due to the inhibitory actions of Semaphorin 3A genetically susceptible individuals. For example, recent studies identifi ed on oligodendrocyte precursor cell diff erentiation.13 Another potential that polymorphisms in Mer Tyrosine Kinase and Tyrosine Kinase 2 therapeutic approach is activation of Retinoid Acid Receptor-, which increase the risk of developing MS.2,3 It has also been suggested that has been shown to promote the development of neural precursors and the destruction of myelin involves an infectious agent or virus, but this induce remyelination in rats and mice.14 4 theory is controversial. Although the initiating factor remains enigmatic, References a central role for CD4+ T cells in disease pathogenesis is fi rmly established. 1. Faraco, G. et al. (2010) Mol. Med. 17:442. Specifi cally, a disturbance in regulatory T cell (Treg) function is thought to underlie the changes in self tolerance that occur during MS.5 Treg cells 2. Couturier, N. et al. (2011) Brain 134:693. are responsible for maintaining immune homeostasis by suppressing 3. Ma, G.Z. et al. (2011) PLoS One 6:e16964. immune responses and preventing autoimmune destruction. 4. Zandian, M. et al. (2011) PLoS One 6:e16820. Studies using the experimental autoimmune encephalomyelitis (EAE) 5. Venken, K. et al. (2010) Trends Mol. Med. 16:58. model of MS support a preventative role for Treg cells. In the presence 6. Kohm, A.P. et al. (2002) J. Immunol. 169:4712. of autoreactive CD4+ T cells, adoptive transfer of Treg cells, or the pharm- 7. Zhang, R. et al. (2011) J. Mol. Neurosci. 44:31. acological expansion of the Treg cell population, attenuated pathology in EAE mice.6,7 However, EAE mice only model one aspect of a multifactorial 8. Gandi, R. et al. (2010) J. Neuroimmunol. 221:7. human disease. In healthy human subjects, the diff erentiation of Treg cells 9. Bailey, S.L. et al. (2007) Nat. Immunol. 8:172. is promoted by immature dendritic cells (DC). In contrast, the increased 10. Menge, T. et al. (2008) Drugs 68:2445. number of activated mature DCs present in MS patients promote 11. Cohen, J.A. et al. (2010) N. Engl. J. Med. 362:402. abnormal diff erentiation of naïve T lymphocytes toward pro-infl ammatory 12. Kuehn, B.M. et al. (2011) JAMA 305:871. Th1 and Th17 subtypes.8 Following activation by DCs in the periphery, autoreactive Th1 and Th17 T helper cells infi ltrate the CNS, recruit micro- 13. Syed, Y.A. et al. (2011) J. Neurosci. 31:3719. glia and macrophages, and promote myelin destruction and neuro- 14. Huang, J.K. et al. (2011) Nat. Neurosci. 14:45.

IL-4, IL-25, IL-33, TSLP TGF-E, IL-2 o TCR Mature DC IMMUNOSUPPRESSION Treg & NEUROPROTECTION IL-12, IL-18, IL-27, IFN- MHCII n Th2 TGF-E J FoxP3 IL-10 GATA-3 IL-4 Th1 Naïve T Cell p T-bet IFN-J IL-10 TGF-E, IL-1E, IL-6, IL-21, IL-23 TGF-E

IL-4 IFN-J AUTOIMMUNITY t Th17 & AXON DESTRUCTION IL-17 Immature DC IL-17 RORJt IL-22 Microglia r q IL-22 s Macrophage

Figure 29. T Cell Diﬀ erentiation and the Pathogenesis of Multiple Sclerosis. A summary of key events during the pathogenesis of MS, including the introduction of an unknown initiating factor to DCs in the periphery , antigen presentation to naïve CD4+ cells , the diff erentiation of naïve T cells toward pro-infl ammatory Th1 and Th17 cells , the release of pro-infl ammatory cytokines , the infi ltration of innate immune cells and further myelin destruction , and the presentation of novel myelin derived antigens and epitope spreading . Researchers are investigating mechanisms to protect vulnerable neurons by shifting T cell diff erentiation toward the generation of immunosuppressive Th2 and Treg cells . 56 For research use only. Not for use in diagnostic procedures. NEURODEGENERATIVE DISEASES

R&D Systems Products for Multiple Sclerosis RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

IL-6 H M R P Ca CR E H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M R Ca Nogo-B H (IHC, WB) M (IHC, WB) F R (B/N, ELISA, IHC, WB) Ca (B/N, ELISA, FC, IHC, WB) CR F P CR (B/N, WB) E (B/N, IHC, WB) F (B/N, IHC, WB) Nogo-C H (IHC, WB) P (B/N, ELISA, FC, IHC, WB) OMgp H M H (WB) M (WB) IL-6 R H M H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M PLP H (IHC) IL-17 H M Ca H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, WB) H M PSMA/FOLH1/ H M H (FC, IHC, WB) IL-17 R H M H (B/N, ELISA, FC, WB) M (B/N, FC, WB) H NAALADase I

IL-23 H M R H (B/N, WB) M (B/N, ELISA, FC, WB) Ca (WB) F (WB) H M RARα/NR1B1 H (IP, WB)

IL-23 R H M H (FC, WB) M (B/N, FC, WB) RARβ/NR1B2 H (IP, WB)

MAG/Siglec-4a RR (B/N, ELISA, IHC, WB) R RARγ/NR1B3 H (IP, WB)

MBP H (WB) M (WB) R (WB) B (WB) Sphingosine Kinase H M H (IP, WB) 1/SPHK1 MOG H (IHC, WB) M (IHC, WB) Sphingosine Kinase H M Myeloperoxidase/ H M H (IHC, WB) M (IHC, WB) H 2/SPHK2 MPO Thrombomodulin/ H M H (ELISA, FC, IHC, IP, WB) M (FC, IHC, IP, WB) H NF2/Merlin H (IHC, WB) BDCA-3

Nogo-A H R H (IHC, WB) R (IHC, WB)

PBS Myelin 40 (pg/mL) E IL-1

0 0 4 24 72 Time after injection (hours)

IL-2 R in Mouse Spleen. Interleukin-2 Receptor  (IL-2 R) was detected in Detection of IFN- and IL-17 Secretion using the Dual-Color ELISpot Kit. Measurement IL-1 Levels Using the DuoSet® ELISA Development Kit. Wild type immersion-fi xed frozen sections of mouse spleen using a Goat Anti-Mouse IL-2 R Interferon- (IFN-, blue spots) and Interleukin-17 (IL-17, red spots) were secreted mice were injected intraperitoneally with PBS or myelin (25 mg/kg body weight) to Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF2438). The tissue was from mouse splenocytes stimulated with PMA and calcium ionomycin. Cytokine induce the production of infl ammatory cytokines. After the indicated time period, levels stained with the NorthernLights™ 557-conjugated Donkey Anti-Goat IgG Secondary secreting cells were visualized using the Mouse IFN-/IL-17 Dual-Color ELISpot Kit of IL-1 were measured in the peritoneal lavage fl uid using the the Mouse IL-1/IL-1F2 Antibody (Catalog # NL001; red) and counterstained (green). (Catalog # ELD5007). DuoSet ELISA Development Kit (Catalog # DY401). * p<0.05 compared to PBS Control. Adapted from Sun, X., (2010) PLoS ONE 5:e9380. IL-1 IL-6 104 104 E

600 * IL-8 103 103 500 RS

400 TNF-D 102 102 FoxP3 FoxP3 300

1 1 10 10 Concentration (pg/mL) 200

100 100 100 100 101 102 103 104 100 101 102 103 104 0 CD4 CD25 CD40L IFN-J IL-1D IL-1E IL-6 IL-8 IL-17 TNF-D

Detection of Human Treg Cells using Multi-Color Flow Cytometry. Human peripheral blood mononuclear cells (PBMCs) were assessed for FoxP3, CD25, and CD4 expression Detection of Human Serum Cytokine Expression During Inﬂ ammation. Cytokines using antibodies and buff ers included in the Human Regulatory T Cell Multi-Color Flow Cytometry Kit (Catalog # FMC013). Quadrants were set based on isotype controls. were detected in the serum of a subject with systemic lupus erythymatosus using the Mosaic ELISA Human Cytokine Panel 1 (Catalog # MEA001). A representative image of an individual well for the data shown is inset. In addition to the 8 spotted capture antibodies, well images show a Reference Spot (lower rightmost spot in each well). Reference Spots provide a strong positive signal to allow easy visualization of well location for template alignment during data analysis. *Without further sample dilution, the values for IL-8 were above the detectable range.

www.RnDSystems.com/go/MS 57 NEURODEGENERATIVE DISEASES

Diabetic Peripheral Neuropathy Diabetes mellitus is a metabolic syndrome in which patients do not produce axonal transport was reversed by upregulation of the antioxidant enzyme suffi cient levels of, or correctly respond to Insulin. Secreted from -cells in Superoxide Dismutase 2 (SOD2). the islets of Langerhans in the pancreas, Insulin is an essential physiological Neuropathic pain is hypothesized to be caused by neuronal hyper- hormone. Under normal conditions, Insulin is secreted in response to excitability, the generation of inappropriate spontaneous activity and increased blood glucose levels, stimulating cellular glucose uptake and exaggerated responses to peripheral stimuli. Hyperexcitability is believed its subsequent storage as Glycogen. Type 1 diabetes is characterized by to result from peripheral changes in ion channel expression, but thalamic a failure to produce enough Insulin. In contrast, Insulin is released during amplifi cation of pain signals may also be involved.5 Recent studies of type 2 and gestational diabetes, but the body exhibits Insulin resistance and spinal dorsal horn neurons suggest that changes in NFB subcellular fails to respond accordingly. Although the specifi c cause remains unknown, localization, GABA-B Receptor downregulation, and increased mGluR5 type 1 diabetes is believed to result following autoimmune destruction of expression may contribute to hypersensitivity associated with DPN.6-8 pancreatic -cells. Increased risk of developing type 2 diabetes is more commonly associated with lifestyle factors, such as obesity. Another group reported that increased BDNF activity impaired voltage-gated potassium channel expression and induced abnormal In addition to retinopathy, nephropathy, and heart disease, the most hyperexcitability in diabetic rats. This eff ect could be blocked using common complication of diabetes is neuropathy.1 Diabetic peripheral an anti-BDNF antibody or a TrkB tyrosine kinase inhibitor.9 In contrast, neuropathy (DPN) aff ects approximately 50% of patients with diabetes. decreased neurotrophic support by Insulin, Insulin-like Growth Factors This condition is characterized by pain and sensory loss that result from (IGF-I and IGF-II), -NGF, and Neurotrophin-3 is thought to be central to nerve injury. Hallmark features of DPN include reductions in motor and diabetes-associated neurodegeneration.10,11 In addition, increased expres- sensory nerve conduction, and structural changes to peripheral nerves sion of VEGF-A in dorsal root ganglia activated the PI 3-K/Akt pathway and such as axonal degeneration, Schwann cell pathology, demyelination, and protected against STZ-induced mechanical allodynia (pain resulting from endoneurial microangiopathy. Although it is believed to be a multifactorial increased sensitivity to touch).12 pathology involving hyperglycemia, oxidative stress, mitochondrial dysfunction, and loss of calcium homeostasis, the molecular mechanisms References that underlie DPN are poorly understood.2 1. Fernyhough, P. & C. Calcutt (2010) Cell Calcium 47:130. The use of streptozotocin (STZ) to injure pancreatic islet cells and induce 2. Fernyhough, P. et al. (2010) Expert Rev. Endocrinol. Metab. 5:39. diabetes in rodents has been essential for the study of DPN. A recent 3. Urban, M.J. et al. (2010) ASN Neuro. 2:e00040. report described the administration of KU-32, a heat shock protein 90 4. Sharma, R. et al. (2010) PLoS One 5:e13463. (HSP90) inhibitor, to combat DPN-related neurodegeneration.3 These ex- 5. Fischer, T.Z. & S.G. Waxman (2010) Nat. Rev. Neurol. 6:462. periments were designed to upregulate a broad cytoprotective response 6. Berti-Mattera, L.N. et al. (2011) Neurosci. Lett. 490:41. by attenuating HSP90-dependent inhibition of HSP70, an established neuroprotective molecular chaperone. Upregulation of HSP70 prevented 7. Wang, X.L. et al. (2011) Neurosci. Lett. 490:112. Neuregulin-1-induced demyelination of cultured dorsal root ganglia and 8. Li, J.Q. et al. (2010) J. Neurochem. 112:162. 3 reversed sensory and motor defi cits in STZ mice (Figure 30). Additional 9. Cao, X.H. et al. (2010) J. Neurochem. 114:1460. studies using STZ mice showed that hyperglycemia induced p38 MAPK 10. Calcutt, N.A. et al. (2008) Curr. Drug Targets 9:47. activation, Tau phosphorylation, and axonal transport defi cits.4 These studies support a causative role for oxidative stress since impairment of 11. Andreassen, C.S. et al. (2009) Brain 132:2724. 12. Pawson, E.J. et al. (2010) Diabetes 59:509.

KU-32 (i.p.) SSTZTZ (i.p..p.)) VZ + 434 (i.m.) Pancreaeas vInsulin VEGF-A Neuropilin-1 ImpImpaired CaCa2+ homeostasis Neurotrophin lossloss VEGF R2 KU-32 HSP70 Oxidatiative stressss Mitochondrial dysfunction HSF-1 Caveolin-1 HSP90 Hyperglycrglycemiaia Dynactin-2

v JNK Spinal Cordord PI 3-K/Akt

Whitehi matter Gray matter Muscle fibers Dorsal Root Ganglion

Figure 30. Novel Therapeutic Candidates for Diabetic Peripheral Neuropathy. Recent studies investigated potential neuroprotective strategies in the streptozotocin (STZ)-induced rodent model of diabetes. Urban et al. showed that intraperitoneal (i.p.) administration of the HSP90 inhibitor KU-32, led to activation of HSP70 in dorsal root ganglia (DRG), decreased expression of phospho-JNK, and neuroprotection.3 In addition, Pawson et al. reported that intramuscular (i.m.) injection of a plasmid encoding a zinc fi nger activator for VEGF-A increased VEGF-A levels in DRG and protected neurons via the PI 3-K/Akt pathway.12 The complete signaling cascades that underlie these observations are yet to be fully elucidated.

58 For research use only. Not for use in diagnostic procedures. NEURODEGENERATIVE DISEASES

R&D Systems Products for Diabetic Peripheral Neuropathy RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

Akt H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R NGF R/TNFRSF16 H M H (FC, IHC, WB) M (IHC, WB)

Akt1 HH (FC, IHC, WB) M (WB) R (WB) H M R Nitrotyrosine Ms (IHC, WB)

Akt2 H (FC, IHC, WB) M (IHC, WB) R (IHC, WB) H NT-3 HH (B/N, ELISA, IHC, WB) H

Akt3 H (FC, WB) p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M

BDNF HH (ELISA, FC, IHC, WB) H p38 HH (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M R

(IF, WB) (IF, WB) (IF, IHC, WB) (IF, WB) (IF, WB) (IHC, WB) (IHC, WB) (IHC, WB) (IP) GABAB R2 H M R B Ca p38 H M R Ms H M Ch (IF, WB) Pr (IF, WB) X (IF, WB) Z (IF, WB) p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M R GLP/EHMT1 H (IHC, IP, WB) M (IHC, IP, WB) p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M GLP-1R H (FC, IHC) PARP H (IP, WB) M (IP, WB) Ms (IHC, WB) Ms GLP-2R H (FC, IHC) PI 3-Kinase p110 H (WB) HSP10/EPF H (WB) M (WB) PI 3-Kinase p110 H (WB) HSP20/HSPB6 H (WB) M (WB) R (WB) PI 3-Kinase p110 H (WB) HSP27 HH (WB) M (WB) R (WB) H M R PI 3-Kinase p55 H (WB) M (WB) R (WB) HSP40/DNAJB1 H (WB) M (WB) R (WB) PI 3-Kinase p85 H (WB) M (WB) R (WB) HSP60 H (IHC, WB) M (IHC, WB) R (IHC, WB) H PI 3-Kinase p85 H (WB) HSP70/HSPA1A H (IHC, WB) M (IHC, WB) R (WB) H M R Potassium Channel R (IHC, WB) X (IHC, WB) HSP90 H (WB) M (WB) R (WB) Kv2.2

IGF-I H M R H (B/N, ELISA, IHC, WB) M (B/N, ELISA, IHC, WB) H M R Potassium Channel M (IHC, WB) R (IHC, WB) Kv3.1 IGF-I R H M H (B/N, ELISA, FC, IHC, WB) M (B/N, FC, IHC, WB) H RAGE H M R Ca H (B/N, ELISA, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M R Ca IGF-II H M H (B/N, IHC, WB) M (B/N, ELISA, IHC, WB) M R (B/N, ELISA, IHC, WB) Ca (ELISA, WB) IGF-II R HH (B/N, ELISA, FC, IHC, WB) H SOD (Superoxide Ms Dismutase) JNK H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R SOD1/Cu-Zn SOD H (IHC, WB) M (WB) R (WB) JNK1 MH (IHC, WB) M (IHC, WB) R (IHC, WB) SOD2/Mn-SOD H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R JNK1/JNK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) SOD3/EC-SOD H (WB) M (WB) R (WB) JNK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R Tau Ms (IHC, WB) mGluR5 H (FC, IHC, WB) M (IHC, WB) R (IHC, WB) TNF- H M R P B Ca CR H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M R P B Ca mGluR5/1a H (IHC, WB) M (IHC, WB) R (IHC, WB) E F Rb RM R (B/N, ELISA, IHC, WB) B (ELISA, IHC, WB) CR E F Pr Rb Neuregulin-1/NRG1 HH (B/N, ELISA, IHC, WB) H Ca (B/N, ELISA, IHC, WB) CR (B/N, WB) E (B/N, IHC, WB) RM GP (B/N, ELISA, IHC, WB) Neuregulin-3/NRG3 M (IHC, WB) TrkB H M H (FC, IHC, WB) M (B/N, IHC, WB) H NFB1 H (ChIP, WB) M (ChIP, WB) H VEGF H M R Ca F Z H (B/N, ELISA, FC, IF, IHC, WB) M (B/N, ELISA, IHC, WB) H M R Ca NFB2 H (ChIP, IHC, WB) M (WB) H R (B/N, ELISA, IHC, WB) Ca (B/N, ELISA, IHC, WB) Z (B/N, WB)

-NGF H M R H (B/N, ELISA, IHC, WB) R (B/N, E, IHC, WB) H R Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Ze b r a fi s h Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot

DCTN1 in Alzheimer’s Disease Brain. Dynactin subunit 1 (DCTN1) was detected Glucagon in the TC-6 Mouse Cell Line. Glucagon was detected in immersion- HSPH1 in Rat Cerebellum. Heat Shock Protein H1 (HSPH1) was detected in perfusion- in immersion-fi xed paraffi n-embedded sections of human Alzheimer’s disease fi xed TC-6 mouse beta cell insulinoma cells using a Mouse Anti-Human/Mouse fi xed frozen sections of rat brain using a Goat Anti-Human/Mouse/Rat HSPH1 Antigen brain using a Mouse Anti-Human/Mouse/Rat Dynactin Subunit 1/DCTN1 Antigen Glucagon Monoclonal Antibody (Catalog # MAB1249). The cells were stained with Affi nity-purifi ed Polyclonal Antibody (Catalog # AF4029). The tissue was stained with Affi nity-purifi ed Polyclonal Antibody (Catalog # AF6657). Before incubation with the NorthernLights™ 557-conjugated Donkey Anti-Mouse IgG Secondary Antibody the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS008; brown) and the primary antibody, the tissue was subjected to heat-induced epitope retrieval (Catalog # NL007; red) and counterstained with DAPI (blue) counterstained with hematoxylin (blue). Specifi c staining was localized to the cell using Antigen Retrieval Reagent-Basic (Catalog # CTS013). The tissue was stained bodies and dendrites of Purkinje neurons. using the Anti-Mouse HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS002; brown) and counterstained with hematoxylin (blue). Specifi c staining was localized to the cytoplasm of hippocampal neurons. www.RnDSystems.com/go/DPN 59 NEURODEGENERATIVE DISEASES

Neuroinﬂ ammation Infl ammation in the brain is induced by activated microglia and reactive that damages neurons and compromises brain function. Chronic astrocytes. Because glial cells are known to exert both neuroprotective neuroinfl ammation is an important research fi eld due to its central role in and neurodegenerative eff ects, their role in neuroinfl ammation prevalent neurodegenerative diseases such as Alzheimer’s disease (AD), represents a major research focus. Microglia are responsible for immune Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and multiple surveillance in the brain, they are the resident immunocompetent sclerosis (MS). In general, these conditions are characterized by an initial and phagocytic cells in the CNS. Astrocytes control blood fl ow and acute neuroinfl ammatory response that fails to eliminate the causative extracellular neurotransmitter levels to ensure a local environment that agent. This is followed by a self-propagating cascade of pathogenic events is optimized for neuronal function. In the absence of insult or injury, glial that leads to a chronic neuroinfl ammatory state. The current consensus cells are immunosuppressed by neurotransmitters, neurotrophic factors, is that chronic infl ammation in the CNS actively contributes to disease anti-infl ammatory cytokines, and cell-cell contact through CD200/CD200 progression and is not merely a secondary event. 1,2 receptor interactions. However, following the detection of increasing Because the number of activated microglia increases in the brain during extracellular concentrations of potassium ions, ATP, or adenosine, AD, especially in regions where toxic Amyloid- protein is deposited as microglia and astrocytes are stimulated to release a potent cocktail of pro- senile plaques, many groups have investigated the potential benefi cial infl ammatory mediators. eff ects of nonsteroidal anti-infl ammatory drugs (NSAID).3,4 Recent Under normal physiological conditions, the brain is considered to be mechanistic studies showed that expression of a signal transduction- immunologically privileged. In fact, antigen presentation is actively defective mutant of the Receptor for Advanced Glycation End Products suppressed, glial cells are maintained in a quiescent state, and immune (RAGE) attenuated IL-1 and TNF- production, and neuroinfl ammation components are excluded from the brain by the blood-brain barrier.1 in a mouse model of AD.5 Using a rat 6-hydroxydopamine lesion model However, neuronal injury, or the presence of toxic material, stimulates the of PD, Walsh et al. reported that microgliosis occurred before release of pro-infl ammatory mediators that induce a neuroinfl ammatory dopaminergic neuron loss in the substantia nigra, supporting the response. When neuroinfl ammation occurs at the right time for an hypothesis that neuroinfl ammation directly eff ects disease progression.6 appropriate extent, it eliminates the triggering insult and restores brain Signaling through Toll-like receptors (TLR) has recently been identifi ed as homeostasis and function. Benefi cial acute neuroinfl ammatory responses a candidate mechanism of neuroinfl ammation during neurodegenerative repair existing damage and minimize further injury. disease. For example, the expression of TLR2 and TLR4 was reported to be 7,8 In contrast, prolonged unregulated release of pro-infl ammatory cytokines, increased on reactive glial cells in spinal cord from ALS patients. such as TNF-, IL-1, and IFN-, creates a detrimental neurotoxic milieu MS-related studies have attempted to unravel the relationship between neurotransmitters, cells of the immune system, and neurodegeneration. Under infl ammatory conditions, excessive concentrations of glutamate are found in the brain, where TNF- has been shown to increase glutamate MOG Glutamate uptake by activated microglia.9 A recent report suggested that glutamate TLR2 may inhibit neuroinfl ammation during MS by reducing the generation Adenylyl Cyclase mGluR4 of IL-17 producing T helper (Th17) cells, an action that is dependent on Gi/o ATP cAMP dendritic cell expression of metabotropic glutamate receptor 4 (mGluR4, Figure 31).10 An additional study reported that low concentrations of PKA glutamate reduced Nitric Oxide Synthase activity in cultured microglia IL-27 11 PKA IL-6, IL-23 by modulating adenosine adenosine A2A receptor signaling. In the experimental autoimmune encephalomyelitis (EAE) model of MS, increased Dendritic Cell IL-23 GABAergic activity potently ameliorated demyelination by decreasing the response of antigen-presenting cells to myelin.12 IL-6 TGF-E References

1. Chang, R.C. et al. (2009) Cell. Mol. Immunol. 6:317.

2. Lyons, A. et al. (2009) Brain Behav. Immun. 23:1020. RORJt IL-17 Th17 DIFFERENTIATION 3. Edison, P. et al. (2008) Neurobiol. Dis. 32:412.

4. Heneka, M.T. et al. (2011) Curr. Alzheimer Res. 8:115.

Microglia IL-17 5. Fang, F. et al. (2010) FASEB J. 24:1043. 6. Walsh, S. et al. (2011) Neuroscience 175:251.

7. Casula, M. et al. (2011) Neuroscience 179:233. Axon Myelin 8. Harada, K. et al. (2011) J. Neurochem. 116:1138.

9. Persson, M. et al. (2005) GLIA 51:1111. Figure 31. Glutamate Exerts Anti-inﬂ ammatory Eﬀ ects via mGluR4. Infl ammatory stimuli, such as myelin oligodendrocyte 10. Fallarino, F. et al. (2010) Nat. Med. 16:897. glycoprotein (MOG) in the EAE model of MS, induce upregulation of IL-6 and IL-23 in activated dendritic cells. Together with TGF- and IL-21, IL-6 and IL-23 promote the diff erentiation and expansion of pro-infl ammatory Th17 cells, which secrete IL-17 11. Dai, S.S. et al. (2010) J. Neurosci. 30:5802. and induce myelin degeneration. Glutamate inhibits the activity of Adenylyl Cyclase and its downstream mediator PKA through 10 12. Bhat, R. et al. (2010) Proc. Natl. Acad. Sci. USA 107:2508. mGluR4 and Gi/o, suppressing Th17 cell diff erentiation and favoring the generation of anti-infl ammatory regulatory T cells (Tregs).

60 For research use only. Not for use in diagnostic procedures. NEURODEGENERATIVE DISEASES

R&D Systems Products for Neuroinﬂ ammation RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs MOLECULE PROTEINS ANTIBODIES ELISAs

CCL21/6Ckine H M H (B/N, ELISA, IHC, WB) M (B/N, ELISA, IHC, WB) H M IL-10 R H M H (B/N, FC, IHC, WB) M (B/N, WB)

CCR2 H (FC) M (FC) IL-10 R HH (B/N, FC, WB) M (FC, WB)

CCR5 H (B/N, FC, IHC, IP, WB) M (FC, WB) R (FC) IL-17 H M Ca H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, WB) H M

CD200 H M H (B/N, ELISA, FC, IHC, WB) M (FC, IHC, WB) H IL-18/IL-1F4 H M R P F RM H (B/N, ELISA(Det), IP, WB) M (B/N, ELISA, IP, WB) H M R (B/N, WB) Ca (IHC, WB) F (B/N, WB) P (IHC, WB) CD200 R1 H M H (B/N, FC, IHC, WB) M (FC, IHC, WB) RM (B/N, IHC, WB)

Chitinase 3-like 3/ MM (WB) M IL-18 R/IL-1 R5 HH (B/N, FC, IHC, WB) M (B/N, FC, WB) ECF-L IL-18 R/IL-1 R7 HH (B/N, FC, WB) M (WB) COX-1 H (FC, WB) M (WB) IL-23 H M R H (B/N, WB) M (B/N, ELISA, FC, WB) Ca (WB) F (WB) H M COX-2 H (FC, IHC, WB) M (FC, IHC, WB) H M IL-23 R H M H (FC, WB) M (B/N, FC, WB) CXCR3 H (B/N, FC, IHC) M (FC) Integrin M/CD11b H (FC, IHC, WB) M (CD, FC, IHC, IP) CXCR4 H (B/N, FC, IHC) M (B/N, FC, IHC) F (FC, IHC) CXCL10/IP-10/CRG-2 H M CR H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, IHC, WB) H M CX3CL1/Fractalkine H M R H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, WB) H M R CR (B/N, WB) R (B/N, ELISA, IHC, WB) CCL2/JE/MCP-1 H M R Ca H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, WB) H M Ca GFAP H (IHC, WB) Ca (B/N, ELISA, IHC, WB) CR (WB)

CXCL2/GRO/MIP-2/ H M R CR H (B/N, FC, WB) M (B/N, ELISA, IHC, WB) R (B/N, ELISA, WB) M R CCL3/MIP-1 H M R CR H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M CINC-3 CR (B/N, WB)

ICAM-1/CD54 H M R H (B/N, ELISA, FC, IHC, IP, WB) M (B/N, ELISA, FC, IHC, WB) H M R CCL4/MIP-1 H M R Ca CR H (B/N, ELISA, FC, IHC, WB) M (B/N, IHC, WB) R (WB) H M R (B/N, ELISA, FC, IHC, WB) Ca (IHC, WB) CR (B/N, WB)

IFN- H M R P B Ca CR H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M R P B Ca Nrf2 H (WB) M (WB) R (WB) E F RM R (B/N, ELISA, IHC, WB) B (ELISA, FC, IHC, WB) CR E F Pr Ca (B/N, ELISA, IHC, WB) CR (B/N, ELISA) Prostaglandin E2/ Ms E (B/N, ELISA, IHC, WB) F (B/N, WB) P (B/N, FC, IHC, WB) PGE2 RM (B/N, ELISA, WB) CCL5/RANTES H M Ca CR F H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, IHC, WB) H M F IL-1/IL-1F1 H M R P CR H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, IHC, IP, WB) H M R Ca (B/N, IHC) CR (B/N, WB) F (B/N, ELISA, WB) R (B/N, ELISA, WB) CR (B/N, WB) P (B/N, WB) CXCL12/SDF-1 H M F RM H M IL-1/IL-1F2 H M R P Ca CR E H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, IP, WB) R H M R P F F RM (B/N, ELISA, IHC, WB) Ca (B/N, FC, WB) CR (B/N, WB) CXCL12/SDF-1 H F H (B/N, ELISA, WB) E (B/N, IHC, WB) F (B/N, ELISA, IHC) P (B/N, ELISA, WB) CXCL12/SDF-1 H IL-1ra/IL-1F3 H M R P E H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, WB) H M E CXCL12/SDF-1 H M F RM H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M P (B/N, IHC, WB) E (ELISA, IHC, WB) TLR1 H M H (FC, WB) M (FC, WB) IL-1 RI H M R H (B/N, ELISA, FC, WB) M (B/N, FC, IHC, WB) H TLR2 H M H (B/N, ELISA, FC, WB) M (FC, WB) H IL-1 RII H M H (B/N, ELISA, FC, IHC, WB) M (FC, IHC, WB) H TLR3 H M H (FC, WB) M (FC, WB) IL-1 RAcP/IL-1 R3 HH (FC, WB) TLR4 HH (B/N, FC, IHC, WB) M (FC) IL-2 H M R P B Ca CR H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, WB) H M R B Ca E E F R (B/N, ELISA, IHC, WB) P (B/N, FC, IHC, WB) B (ELISA, IHC, WB) F TLR4/MD-2 Complex H Ca (B/N, ELISA, IHC, WB) CR (B/N, WB) E (ELISA) TLR5 H (FC, IHC) IL-2 R H M R Ca H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M R (FC, IHC, WB) Ca (FC) TLR6 MM (FC, WB)

IL-2 R HH (B/N, FC, IHC, WB) M (FC, WB) TLR7 H (FC) M (WB) R (WB)

IL-6 H M R P Ca CR E H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M R P Ca CR TLR9 H (FC) F R (B/N, ELISA, IHC, WB) Ca (B/N, ELISA, FC, IHC, WB) F CR (B/N, WB) E (B/N, IHC, WB) F (B/N, IHC, WB) TLR10 H (WB) P (B/N, ELISA, FC, IHC, WB) TNF- H M R P B Ca CR H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M R P B Ca IL-6 R H M H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M E F Rb RM R (B/N, ELISA, IHC, WB) B (ELISA, IHC, WB) CR E F Pr Rb Ca (B/N, ELISA, IHC, WB) CR (B/N, WB) E (B/N, IHC, WB) RM CXCR1/IL-8 RA H (B/N, FC, IHC) GP (B/N, ELISA, IHC, WB)

CXCR2/IL-8 RB H (B/N, FC, IHC) M (B/N, FC) TNF-/TNFSF1 H M H (B/N, ELISA, FC, IHC, WB) M (IHC, WB) H

CXCL8/IL-8 H P Ca F H (B/N, ELISA, FC, IHC, WB) Ca (B/N, ELISA, IHC, WB) H P Ca F TNF RI/TNFRSF1A H M Ca H (B/N, ELISA, FA, FC, IHC, WB) H M F (B/N, ELISA, IHC, WB) P (B/N, ELISA, WB) M (B/N, ELISA, FA, FC, IHC, IP, WB)

IL-10 H M R P Ca CR E H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M R P Ca E TNF RII/TNFRSF1B H M H (B/N, ELISA, FC, IHC, WB) M (ELISA, FC, IHC, IP, WB) H M F V R (B/N, ELISA, IHC, WB) Ca (B/N, ELISA, WB) CR (WB) F E (B/N, ELISA, IHC, WB) P (B/N, ELISA, IHC, WB)

www.RnDSystems.com/go/Neuroinﬂ ammation 61 NEURODEGENERATIVE DISEASES

Autophagy & the Ubiquitin Proteasome System In neurons, protein degradation promotes cellular homeostasis and Ubiquitination tags proteins for ATP-dependent non-lysosomal degrad- regulates the quality of the expressed proteins. Nuclear and cytoplasmic ation in the proteasome.4 This process requires sequential reactions by proteins are typically destroyed by the ubiquitin proteasome system Ubiquitin (Ub)-activating (E1), Ub-conjugating (E2), and Ub-ligase (E3) (UPS), whereas larger protein complexes, membrane-associated proteins, enzymes. The human genome encodes approximately two E1, fi fty E2, and cellular organelles are hydrolyzed in the lysosomal compartment by a and fi ve hundred E3 enzymes, suggesting that the combination of E2 catabolic process termed autophagy. There are three forms of autophagy: and E3 determines substrate specifi city. Once ubiquitinated, substrates macroautophagy, microautophagy, and chaperone-mediated autophagy are conveyed to the proteasome by E3 enzymes, shuttling factors, or by (CMA).1 During macroautophagy, the most common form, regions of the binding to Ub receptors within the proteasomal membrane.5 Importantly, cytosol are enveloped by autophagosomes, double membrane sequest- the presence of approximately one hundred deubiquitinating enzymes ering vesicles. The inner vesicle, or autophagic body, is subsequently (DUBs) ensures that UPS-mediated degradation is a dynamic process. The delivered to the degradative compartment of the lysosome where activity of DUBs can promote UPS activity by ensuring the availability hydrolytic enzymes breakdown protein components for recycling. of free Ub. In contrast, DUB activity antagonizes protein destruction if Autophagic dysfunction has been linked to several neurological deubiquitination occurs prior to proteasome delivery. disorders that are characterized by defective protein clearance including Like autophagy, UPS dysfunction is thought to contribute to the Alzheimer’s disease (A and Tau), Parkinson’s disease (-Synuclein), development of neurodegenerative disease.4 In addition, Ub-mediated Huntington’s disease (Huntingtin), and amyotrophic lateral sclerosis signaling has been shown to play a critical role during long-term synaptic (TDP-43).1 The detrimental changes in autophagy associated with these plasticity and neurodevelopment. Studies have identifi ed many Ub conditions may refl ect decreased induction, increased repression, enzymes that regulate synaptic function (i.e. Nedd4, Parkin, UCH-L1) and altered cargo recognition, ineffi cient membrane fusion, and/or impaired neuronal activity is known to modulate the subcellular localization of degradation. This posited association has presented the possibility the proteasome.6 For example, Bingol et al. reported that NMDA receptor that pharmacological stimulation of autophagy may represent a novel dependent increases in intracellular calcium levels caused proteasome therapeutic strategy for neurodegenerative disease. Potential molecular organelles to shift from the dendritic shaft to the spine, an event that targets for the induction of autophagy include Beclin-1 and mTOR. Beclin-1 was dependent on the activation of CaMKII.7 Moreover, recent studies is required for the nucleation of the phagophore, the isolation membrane demonstrated that Ub modifi cation regulates neuronal excitability via the of the autophagosome, at discrete regions of the endoplasmic reticulum degradation of L-type voltage-gated calcium channel subunit Cav1.2 and called omegasomes.2 In contrast, inhibition of mTOR (mammalian target the AMPA receptor subunit GluR1.8,9 During development, local protein of rapamycin), a negative regulator of autophagy, has been shown to slow degradation by the UPS in growth cones is thought to contribute to neurodegeneration in experimental models of Huntington’s disease.3 axon guidance.10 Equally, the UPS aff ects dendrite pruning and axonal branching.11,12 Studies in Xenopus retinal ganglion cells indicated that the E3 ligase NEDD4 regulates axonal branching by controlling the expression 13 TNIK of the phosphatase PTEN in growth cones (Figure 32). NEDD4 TNIK Target Neuron Rap2A DENDRITE References Ub ABORIZATION Rap2A 1. Wong, E. & A.M. Cuervo (2010) Nat. Neurosci. 13:805. Ub Ub MINK 2. Zhong, Y. et al. (2009) Nat. Cell. Biol. 11:468.

3. Ravikumar, B. et al. (2004) Nat. Genet. 36:585.

4. Bingol, B. & M. Sheng (2011) Neuron 69:22. DCC Homodimer 5. Glickman, M.H. & D. Raveh (2005) FEBS Lett. 579:3214.

6. Hegde, A.N. (2010) Learn. Mem. 17:314. Netrin Ca2+ 7. Bingol, B. et al. (2010) Cell 140:567.

8. Altier, C. et al. (2011) Nat. Neurosci. 14:173. TRPC1 9. Fu, A.K. et al. (2011) Nat. Neurosci. 14:181.

10. Campbell, D.S. & C.E. Holt (2001) Neuron 32:1013. uCa2+ AXON BRANCHING 11. Kuo, C.T. et al. (2006) Neuron 51:283. Growth Cone NEDD4 12. Drinjakovic, J. et al. (2010) Neuron 65:341. GSK-3 UbUbUb NEDD4 E PTEN 13. Kawabe, H. et al. (2010) Neuron 65:358. ✗ PI 3-K Proteasomal degradation PROTEIN mTOR TRANSLATION

Figure 32. Neurodevelopmental Eﬀ ects of the E3 Ligase NEDD4. Recent studies showed that NEDD4 downregulates PTEN to mediate PI 3-K-dependent axon branching.12 Protein translation stimulated by the activation of mTOR may also contribute to cytoskeletal changes. In addition, monoubiquitination of Rap2A by NEDD4 was shown to promote dendrite formation.13

62 For research use only. Not for use in diagnostic procedures. NEURODEGENERATIVE DISEASES

R&D Systems Products for Autophagy & the Ubiquitin Proteasome System RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Autophagy Ras H (WB) M (WB) R (WB)

Akt1 HH (FC, IHC, WB) M (WB) R (WB) H M R Rheb H (IHC, WB) M (IHC, WB) R (IHC, WB)

AMPK1/2 H (WB) TOR H (FC, IHC, IP, WB) M (FC, IHC, IP, WB) R (IP, WB) H

AMPK1 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R E1 Ubiquitin/Ubl Activating Enzymes & Inhibitors

AMPK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) ISG15 Activating HH Enzyme/UBE1L AMPK1 H (IHC, WB) M (IHC, WB) R (IHC, WB) NEDD8 Activating H AMPK2 H (WB) M (WB) Enzyme (APPBP1/ UBA3) ATG3/Apg3L/Apg3p HH (WB) M (WB) R (WB) PYR 41 Ms ATG4A H (IP, WB) SUMO Activating H Y ATG4B/Apg4b HH (IP, WB) Enzyme E1 (SAE1/ ATG5 H (IHC, WB) M (IHC, WB) R (IHC, WB) UBA2) ATG7 H Ubiquitin E1 H Enzyme/UBEL2 ATG10 H (WB) Ubiquitin-activating H Rb Y ATG12 H (WB) M (WB) Enzyme/UBE1

Bcl-2 HH (B/N, IHC, IP, WB) M (IHC, IP, WB) R (IHC, IP, WB) H UFM1 Activating H Enzyme/UBA5 (IHC, IP, WB) (IHC, IP, WB) (IHC, IP, WB) Bcl-xL H M H M R H M E2 Ubiquitin/Ubl Conjugating Enzymes Beclin 1/ATG6 H (WB) M (WB) R (WB) HR6A/UBE2A H BNIP3 H (WB) HR6B/UBE2B H BNIP3L H (WB) M (WB) R (WB) UbcH (E2) Enzyme H eIF2 H (WB) Set ERK1/ERK2 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R UbcH2/UBE2H HH ERK1 HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R UbcH3 HH ERK2 HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R UbcH5a/UBE2D1 H FoxO3 H (WB) M (WB) UbcH5b/UBE2D2 H GABARAP/Apg8p1 H UbcH5c/UBE2D3 H GATE-16/Apg8p2 H UbcH6/UBE2E1 HH HIF-1 H (ChIP, FC, IHC, IP, WB) M (ChIP, FC, IHC, IP, WB) H M UbcH7 HH R (ChIP, IP, WB) UbcH8 H LC3/MAP1LC3A/ H Apg8p3 UbcH10/UBE2C HH

LKB1 H (WB) UbcH5/UBE2D H

MEK1/MEK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R UBE2E3 H

MEK1 H (IHC, WB) M (IHC, WB) R (IHC, WB) B (WB) Ca (WB) UBE2F H Ch (WB) Pr (WB) X (WB) UBE2G2 H MEK2 HH (IHC, WB) M (IHC, WB) R (IHC, WB) UBE2I/Ubc9 HH p27/Kip1 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H UBE2K/E2-25K HH (IHC, WB) M (IHC, WB) R (WB) p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M UBE2M/Ubc12 HH p38 HH (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M R UBE2N/Ubc13 H p53 HH (ChIP, FC, IHC, IP, WB) M (ChIP, IP, WB) R (ChIP, IP, WB) H M UBE2N (Ubc13)/ H p70 S6 Kinase HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R Uev1a Complex

PERK H (WB) UBE2S H

PINK1 H (IHC) UBE2T H

PP2A H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R Uev1a (Mms2)/ H UBE2V1 PTEN HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R Ufc1 H Raf-1 HH (IHC, WB) M (IHC, WB) R (IHC, WB) B (WB) Ca (WB) Ch (WB) Pr (WB) Use1/UBE2Z H * UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot

www.RnDSystems.com/go/Autophagy 63 NEURODEGENERATIVE DISEASES

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* E3 Ubiquitin Ligases & Inhibitors Proteasome A20/TNFAIP3 H 19S Proteasome H

BRCA1 H (IHC, IP, WB) M (IHC, WB) R (IHC, WB) 20S H M R Ca Immunoproteasome CBL H (WB) M (WB) 20S Proteasome H M R Ca Rb Rb Ms GRAIL/RNF128 H (WB) 26S Proteasome H Ms Rb HLI 373 Ms Ac-Arg-Leu-Arg-AMC Ms MDM2/HDM2 H (IHC, IP, WB) M (IHC, WB) R (IHC, WB) H (Ac-RLR-AMC) MuRF1/TRIM63 HH (WB) M (WB) R (WB) H Ac-Leu-Leu-Met- Ms CHO (Ac-LLM-CHO) NEDD4 H (WB) M (WB) R (WB) NSC 146109 Ms Ac-Leu-Leu-Nle-CHO Ms Hydrochloride (MG-101) NSC 66811 Ms AM 114 Ms Boc-Leu-Arg-Arg- Ms Parkin HH (IHC, WB) H AMC (Boc-LRR-AMC) RNF2 H (WB) M (WB) R (WB) Clasto-Lactacystin Ms RNF8 H (WB) -Lactone

Skp2 H (WB) Epoxomicin Ms

SMURF2 H (WB) M (WB) R (WB) Gliotoxin Ms

STUB1/CHIP H (WB) IU1 Small Molecule Ms Inhibitor of USP14/ Thalidomide Ms SMI-USP14

TRIM21 H (IHC, WB) Lactacystin Ms

(WB) (WB) TRIM32 H M Me4BodipyFL- Ms Ahx Leu VS UBR5 H (WB) M (WB) R (WB) 3 3 PA28 Activator  H Deubiquitinating Enzymes (DUBs) Subunit AMSH/STAMBP H (WB) PA28 Activator  H Ataxin UIM Domains H Subunit PA28 Activator  H Ataxin-3 H Subunit BAP1 H S5a/Angiocidin H Ms M (WB) Deconjugating H Suc-Leu-Leu-Val-Tyr- Ms Enzyme Set AMC (Suc-LLVY-AMC) Isopeptidase T/USP5 H Rb Suc-Leu-Tyr-AMC Ms LDN 57444 Ms (Suc-LY-AMC) NEDP1/SENP8 H Z-Leu-Leu-Glu-AMC Ms (Z-LLE-AMC) NSC 632839 Ms Hydrochloride Z-Leu-Leu-Leu-AMC Ms (Z-LLL-AMC) Otubain-1 H Z-Leu-Leu-Leu-CHO Ms SUMO1-Specifi c H (MG-132) Peptidase 1/SENP1 Z-Leu-Leu-Phe-CHO Ms SUMO1-Specifi c H (Z-LLF-CHO) Peptidase 2/SENP2 Z-lle-Glu(OtBu)-Ala- Ms UBP43/USP18 H Leu-CHO (Z-IE-OtBu-AL-CHO) UCH-L1 H M H (IHC, IP, WB) M (WB) R (WB) Ubiquitin UCH-L3 H Rb H (WB) M (WB) R (WB) Ubiquitin H Pz Pl Y Ms H (IHC, WB) H Rb Ms USP2 HM (WB) Ubiquitin/ H (IHC, WB) USP7 H Ubiquitin+1

USP8 H Ubiquitin+1 HH (ELISA, IHC, WB) H USP14 H Di-Ubiquitin/Ub2 H Ms

64 For research use only. Not for use in diagnostic procedures. NEURODEGENERATIVE DISEASES

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Tri-Ubiquitin/Ub3 H SUMO1 HH (WB) M (WB) H Tetra-Ubiquitin/Ub4 H SUMO1/2/3 H

Penta-Ubiquitin/Ub5 H SUMO2/3/4 H (WB) M (WB)

Hexa-Ubiquitin/Ub6 H SUMO2 HH (WB) M (WB) H Octa-Ubiquitin/Ub8 H Poly-SUMO2 H Poly-Ubiquitin H Di-SUMO2 H

Ubiquitin-like Modiﬁ ers (UBLs) SUMO3 HH (IHC, WB) M (WB) H FAT10 H Poly-SUMO3 H

ISG15/UCRP HH (IHC, WB) H Ms Di-SUMO3 H

NEDD8 HH (IHC, WB) M (IHC, WB) R (IHC, WB) H Ms SUMO4 H (WB) M (WB)

PIAS2 H (WB) SUMO-interacting Ms Motif (SIM) PIAS3 H (WB) UFM1 HH H Ms SUMO H Ms * UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot

3 ain 2 kat r Br 6 ain u e 5 J HT- L - s kDa Br C u kat r at u J MR R Mo

PTEN NEDD4 2 113

g Lysate) 93 P

65 1 PTEN (ng/40

0 Jurkat MRC-5 Rat Mouse 24 Brain Brain

Measurement of PTEN Levels using the DuoSet® IC ELISA. Phosphatase and Ubiquitin/Ubiquitin+1 in Alzheimer’s Disease Cortex. Ubiquitin/Ubiquitin+1 Detection of Human, Mouse, and Rat NEDD4 by Western Blot. Western blots Tensin Homolog Deleted on Chromosome 10 (PTEN) protein levels in lysates was detected in immersion-fi xed paraffi n-embedded sections of human Alzheimer’s show lysates of the Jurkat human acute T cell leukemia cell line, the HT-2 mouse T prepared from the Jurkat human acute T cell leukemia cell line and the MRC-5 disease brain using a Mouse Anti-Human Ubiquitin/Ubiquitin+1 Monoclonal cell line, and the L6 rat myoblast cell line. The PVDF membranes were probed using a human lung fi broblast cell line, as well as rat and mouse brain, were quantifi ed Antibody (Catalog # MAB701). The tissue was stained with the Anti-Mouse HRP- Mouse Anti-Human/Mouse/Rat NEDD4 Monoclonal Antibody (Catalog # MAB6218) using the DuoSet IC ELISA (Catalog # DYC847, bar graph). The same lysates were DAB Cell & Tissue Staining Kit (Catalog # CTS002; brown) and counterstained with followed by a HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody (Catalog also immunoblotted using a Rabbit Anti-Human/Mouse/Rat PTEN Antigen Affi nity- hematoxylin (blue). Specifi c labeling was localized to the cytoplasm of neurons in # HAF007). A specifi c band was detected for NEDD4 at approximately 118 kDa (as purifi ed Polyclonal Antibody (Catalog # AF847, inset). The DuoSet IC ELISA results the cortex. indicated). correlate well with the relative amounts of total PTEN detected by Western blot.

www.RnDSystems.com/go/Autophagy 65 NEURODEGENERATIVE DISEASES

Apoptosis Apoptosis is the process of programmed cell death. It plays critical roles in development and immunity, as well as cancer and neurodegenerative disease. Apoptosis is initiated by an extrinsic or intrinsic death signal, This specialized catalog features a which ultimately leads to the activation of intracellular caspases (Figure restricted list of products for Apoptosis. 33). Multiple protein families are involved in the signaling pathways For a complete list of related products that promote or inhibit caspase activation. The extrinsic pathway of please visit: caspase activation (green arrows) involves ligand binding to cell surface www.RnDSystems.com/go/Apoptosis death receptors, belonging to the TNF receptor superfamily, while the intrinsic pathway (blue arrows) involves a loss of the integrity of the outer mitochondrial membrane, which is regulated by members of the Bcl-2 family of proteins. In addition to these two families of proteins, numerous other intracellular proteins, kinases, and transcription factors To request a copy of our Apoptosis Catalog play a role in promoting cell death, or acting to prevent it. R&D Systems please visit our website at: has developed and validated a wide variety of research reagents useful for www.RnDSystems.com/Request the characterization of these pro- and anti-apoptotic signaling pathways.

TNF-D Fas Ligand TRAIL TWEAK TNF RI Fas TRAIL R1 TRAIL R2

RIP1 DR3 TRADD FADD FADD (or another TWEAK R) TRADD FADD Pro-caspase-8,-10 Pro-caspase-8,-10 Pro -caspase-8,-10 FADD FADD TRAF-2 Pro-caspase-8,-10 Pro-caspase-8,-10 TRADD FLIP TRADD TRAF-2 RIP1 FADD Pro-caspase-8,-10 14-3-3 Caspase-8,-10 Bad Extrinsic Pathway of Caspase Activation P P P BID Cytochrome c tBID BAK Caspase-3

Bad Bad tBID tBID tBID tBID IAPs Pro-caspase-3 BAK Bax Bax Bad Bad BAK SMAC/Diablo IAPs Bcl-2 HTRA2/Omi Caspase-7 BAK Bcl-xL Target Molecules Bcl-2 (e.g. Actin, Nuclear Lamins, BAK Caspase-9 Pro-caspase-7 ICAD, PARP) Bcl-xL Bax tBID Cytochrome c Caspase-6 Bax tBID APAF-1 Pro-caspase-6 Bcl-2 Pro-caspase-9 Bax Bcl-x Bax HSPs L Bcl-2 Bcl-2 tBID Bax tBID Bax Bad Cytochrome c AIF Apoptosome Cytochrome c 14-3-3 Bax tBID Endo G Bad Bad Bad BID P P P Bad PIDDosome Caspase-2 DNA DAMAGE DNA FRAGMENTATION ✗ ✗ ✗ ✗ Pro-caspase-2 ARC Intrinsic Pathway of Caspase Activation Pro-apoptotic: Bad, Bax, BID, Noxa, PUMA, PIDD, others CRADD/RAIDD P Anti-apoptotic: 14-3-3, p21, others p53 p53 p53 Note: This diagram conveys a general overview of selected processes and should neither be considered comprehensive nor definitive. PIDD The details of these processes are understood to be subject to interpretation.

Figure 33. The Extrinsic and Intrinsic Pathways of Caspase Activation.

66 For research use only. Not for use in diagnostic procedures. NEURODEGENERATIVE DISEASES

R&D Systems Products for Apoptosis RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Apoptosis Adaptor Proteins ASK1 HH (IHC, WB)

14-3-3 H (WB) M (WB) R (WB) B (WB) Ca (WB) Ch (WB) Pr (WB) CaMKK H (WB) R (WB) X (WB) Z (WB) DRAK1 H (WB) 14-3-3 H (WB) M (WB) R (WB) DRAK2 H (WB) M (WB) R (WB) 14-3-3 H (WB) M (WB) R (WB) ERK1/ERK2 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R 14-3-3 H (WB) M (WB) R (WB) ERK1 HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R 14-3-3 H (WB) M (WB) R (WB) ERK2 HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R 14-3-3 H (WB) M (WB) R (WB) GL H (WB) M (WB) R (WB) 14-3-3 H (WB) M (WB) R (WB) GSK-3/ H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R 14-3-3 HH (WB) M (WB) GSK-3 H (IHC, WB) M (IHC, WB) R (IHC, WB) H ASC H (WB) GSK-3 HH (FC, IHC, WB) M (FC, WB) R (FC, WB) H M R BAP31 H (WB) Jak1 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) CD2AP H (WB) M (WB) R (WB) Jak2 M (WB) R (WB) CIDEA H (WB) Jak3 H (FC, WB) CIDEC H (IHC, WB) JNK H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R CRADD H (WB) M (WB) R (WB) JNK1/JNK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) Crk H (WB) M (WB) JNK1 MH (IHC, WB) M (IHC, WB) R (IHC, WB) CrkL H (WB) M (WB) R (WB) JNK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R Daxx H (WB) LMTK2 H (WB) DFF40/CAD H (WB) MAP3K8/Tpl2/COT HH (WB) DFF45/ICAD H (WB) MEK1/MEK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R FADD H (IHC, WB) MEK1 H (IHC, WB) M (IHC, WB) R (IHC, WB) B (WB) Ca (WB) FLIP HH (WB) M (WB) Ch (WB) Pr (WB) X (WB)

MFG-E8 H M H (FC, WB) M (ELISA, WB) M MEK2 HH (IHC, WB) M (IHC, WB) R (IHC, WB)

MyD88 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H MELK H (IHC, WB)

NGFRAP1/BEX3 H (IHC, WB) MKK3/MKK6 H (WB) M (WB) R (WB)

PEA-15 H (WB) M (WB) MKK3 H (IHC, WB) M (IHC, WB) R (IHC, WB) H

RIP1 H (WB) M (WB) R (WB) MKK4 H (IHC, WB) M (IHC, WB) R (IHC, WB)

SHB H (IHC) MKK6 H (IHC, WB) M (IHC, WB) R (IHC, WB) H

SODD/BAG4 H (WB) M (WB) R (WB) MKK7 H (IHC, WB)

STING/TMEM173 H (FC, WB) MLK2 H (WB)

TANK H (WB) M (WB) MLK3 H (WB)

TRADD H (FC, IHC, WB) MLK4 H (IHC, WB) M (IHC, WB) R (IHC, WB)

TRAF-1 H (WB) MSK1/MSK2 H (WB)

TRAF-2 H (WB) M (WB) R (WB) MSK1 H (IHC, WB) M (IHC, WB)

TRAF-3 H (WB) M (WB) R (WB) MSK2 H (IHC, WB) M (IHC, WB)

TRAF-4 H (WB) MST1/STK4 H (WB)

TRAF-6 H (WB) MST2/STK3 H (WB) M (WB) R (WB)

TRAM/TICAM2 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M

TRIF/TICAM1 H (IHC, WB) p38 HH (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M R

Apoptosis Intracellular Kinases p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M

Akt H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M R

Akt1 HH (FC, IHC, WB) M (WB) R (WB) H M R p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M

Akt2 H (FC, IHC, WB) M (IHC, WB) R (IHC, WB) H p70 S6 Kinase HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R

Akt3 H (FC, WB) p70 S6 Kinase  H (WB) * UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat E Equine F Feline Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot

www.RnDSystems.com/go/Apoptosis 67 NEURODEGENERATIVE DISEASES

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Apoptosis Intracellular Kinases, continued MSX1 H (WB) M (WB)

p55  H (WB) M (WB) c-Myc H (ChIP, IHC, WB) H

PDK-1 HH (WB) M (WB) R (WB) NFB1 H (ChIP, WB) M (ChIP, WB) H

PI 3-Kinase p55 H (WB) M (WB) R (WB) NFB2 H (ChIP, IHC, WB) M (WB) H

PI 3-Kinase p85 H (WB) M (WB) R (WB) p53 HH (ChIP, FC, IHC, IP, WB) M (ChIP, IP, WB) R (ChIP, IP, WB) H M

PI 3-Kinase p110 H (WB) PIAS3 H (WB)

PI 3-Kinase p110 H (WB) RelA/NFB p65 H (ChIP, FC, WB) M (ChIP, FC, WB) H

PI 3-Kinase p110 H (WB) RelB H (IHC, WB)

PI 3-Kinase p85 H (WB) c-Rel H (ChIP, IHC, WB) M (ChIP, IHC, WB) H

PIK3R4 H (WB) STAT1 H (FC, IHC, IP, WB) M (FC, IP, WB) H M

PIK3R5 H (WB) STAT2 H (FC, IHC, IP, WB) H

PIM1 H STAT3 H (ChIP, FC, IHC, IP, WB) M (ChIP, FC, IHC, IP, WB) H M R (ChIP, FC, IHC, IP, WB) PINK1 H (IHC) STAT4 H (ELISA, IP, WB) M (ELISA, IP, WB) H PKA C H (WB) M (WB) R (WB) STAT5a/b H (ChIP, FC, IHC, WB) M (ChIP, WB) H M PKA C HH (WB) M (WB) R (WB) STAT5a H (FC, IHC, IP, WB) M (IHC, IP, WB) PKA C/ H (WB) M (WB) R (WB) STAT5b H (FC, IP, WB) M (FC, IP, WB) PKA C HH (WB) M (WB) R (WB) STAT6 H (FC, IHC, IP, WB) M (FC, IP, WB) R (FC, WB) H M PKA RI H (WB) M (WB) R (WB) TRPS1 H (WB) PYK2/FAK2 HH (WB) Autophagy Rictor H (WB) AMPK1/2 H (WB) ROCK1 HH (WB) M (WB) R (WB) AMPK1 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R ROCK2 H (WB) M (WB) R (WB) AMPK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) RSK H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R AMPK1 H (IHC, WB) M (IHC, WB) R (IHC, WB) RSK1/RSK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) AMPK2 H (WB) M (WB) RSK1 HH (IHC, WB) M (IHC, WB) R (IHC, WB) H ATG3/Apg3L/Apg3p HH (WB) M (WB) R (WB) RSK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) ATG4A H (IP, WB) RSK3 H (IHC, WB) M (IHC, WB) ATG4B/Apg4b HH (IP, WB) RSK4 H (WB) ATG5 H (IHC, WB) M (IHC, WB) R (IHC, WB) Src H V H (IHC, WB) M (IHC, WB) R (IHC, WB) H ATG7 H SYK HH (WB) ATG10 H (WB) TOR H (FC, IHC, IP, WB) M (FC, IHC, IP, WB) R (IP, WB) H ATG12 H (WB) M (WB) Tyk2 H (WB) Bcl-2 HH (B/N, IHC, IP, WB) M (IHC, IP, WB) R (IHC, IP, WB) H Apoptosis Transcription Factors & Regulators Bcl-x H M H (IHC, IP, WB) M (IHC, IP, WB) R (IHC, IP, WB) H M C1D H (WB) L Beclin 1/ATG6 H (WB) M (WB) R (WB) Cited-2 H (WB) M (WB) BNIP3 H (WB) E2F-1 H (IHC, WB) BNIP3L H (WB) M (WB) R (WB) E2F-2 H (WB) eIF2 H (WB) E2F-4 H (WB) ERK1/ERK2 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R FosB/G0S3 H (IHC, WB) M (WB) ERK1 HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R FoxO3 H (WB) M (WB) ERK2 HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R FRA-1 H (WB) FoxO3 H (WB) M (WB) IRF6 H (WB) M (WB) GABARAP/Apg8p1 H IRF8 H (IHC, WB) GATE-16/Apg8p2 H IRF9 H (WB) M (WB) HIF-1 H (ChIP, FC, IHC, IP, WB) M (ChIP, FC, IHC, IP, WB) H M JunB H (WB) R (ChIP, IP, WB) JunD H (WB) M (WB) LC3/MAP1LC3A/ H Apg8p3 MAD1L1 H (WB) LKB1 H (WB) Max H (WB)

68 For research use only. Not for use in diagnostic procedures. NEURODEGENERATIVE DISEASES

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

(IHC, WB) (IHC, WB) (IHC, WB) (IHC, IP, WB) (IHC, IP, WB) (IHC, IP, WB) MEK1/MEK2 H M R H M R Bcl-xL H M H M R H M MEK1 H (IHC, WB) M (IHC, WB) R (IHC, WB) B (WB) Ca (WB) BID H M H (IP, WB) M (IP, WB) Ch (WB) Pr (WB) X (WB) BIK H (IHC, WB) MEK2 HH (IHC, WB) M (IHC, WB) R (IHC, WB) (IHC, WB) (WB) BIML HHM p27/Kip1 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H BMF H (IHC) p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M BNIP3 H (WB) p38α HH (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M R BNIP3L H (WB) M (WB) R (WB) p53 HH (ChIP, FC, IHC, IP, WB) M (ChIP, IP, WB) R (ChIP, IP, WB) H M BOK RH (WB) M (WB) R (WB) p70 S6 Kinase HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R Hrk HH (WB) p70 S6 Kinase  H (WB) Mcl-1 HH (IHC, WB) PERK H (WB) Caspases & Regulators PP2A H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R APAF-1 H (WB) PTEN HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R ARC HH (IHC, WB) M (IHC, WB) R (IHC, WB) Raf-1 HH (IHC, WB) M (IHC, WB) R (IHC, WB) B (WB) Ca (WB) Ch (WB) Pr (WB) ASC H (WB)

Ras H (WB) M (WB) R (WB) CARD9 H (WB)

Rheb H (IHC, WB) M (IHC, WB) R (IHC, WB) CARD11/CARMA1 H (WB) M (WB) R (WB)

TOR H (FC, IHC, IP, WB) M (FC, IHC, IP, WB) R (IP, WB) H Caspase Bcl-2 Family Caspase-1 H (IHC, WB) H

Bad H (IHC, WB) M (WB) H Caspase-2 HH (WB) M (WB)

Bag-1 H (IP, WB) M (IP, WB) Caspase-3 HH (IHC, IP, WB) M (IHC, IP, WB) H M

BAK HH (WB) M (WB) R (WB) Caspase-4

Bax H (IHC, IP, WB) M (IHC, IP, WB) R (IP, WB) H Caspase-6 Bax- H Caspase-7 HH (WB) M (WB)

Bcl-2 HH (B/N, IHC, IP, WB) M (IHC, IP, WB) R (IHC, IP, WB) H Caspase-8 HH (WB) M (WB)

Bcl-2 related protein H M M (WB) Caspase-9 H (ELISA, IHC, WB) A1 Caspase-10 HH (IHC, WB) Bcl-10 H (WB) M (WB) R (WB) Caspase-12 M (WB) R (WB) Bcl-w HH (B/N, IHC, WB) M (B/N, IHC, WB) Caspase Peptide Ms Bcl-x H (IP, WB) Inhibitors (IHC, WB) (WB) * UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS cIAP-1/HIAP-2 HHM Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat E Equine F Feline Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot ain Br

Untreated e s rh14-3-3 1 u Pro-Caspase-3Cleaved Caspase-3 3 kDa Mo EHKJVT] A4 94 65

39 Treated Pro-Caspase-3Cleaved Caspase-3 14-3-3V 23 19

Detection of Multiple Apoptosis-related Proteins using the Proteome Proﬁ ler™ TRAIL/TNFSF10 in Human Cortex. TNF-related apoptosis-inducing ligand (TRAIL)/ Detection of 14-3-3 by Western Blot. Western blot shows lysates of the A431 Human Apoptosis Antibody Array. Cell lysates from the Jurkat human acute T TNFSF10 was detected in immersion-fi xed paraffi n-embedded sections of human human epithelial carcinoma cell line and mouse brain tissue. The PVDF membrane cell leukemia cell line, untreated or treated with 1μM staurosporine were assessed brain using a Goat Anti-Human TRAIL/TNFSF10 Antigen Affi nity-purifi ed Polyclonal was probed with a Goat Anti-Human/Mouse/Rat 14-3-3 Antigen Affi nity-purifi ed for the relative levels of thirty- fi ve diff erent apoptosis-related proteins using the Antibody (Catalog # AF375). Before incubation with the primary antibody, the tissue Polyclonal Antibody (Catalog # AF4424) followed by a HRP-conjugated Rabbit Proteome Profi ler Human Apoptosis Antibody Array Kit (Catalog # ARY009). Proteins was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic Anti-Goat IgG Secondary Antibody (Catalog # HAF017). For additional reference, were visualized by chemiluminescent detection. (Catalog # CTS013). The tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue recombinant human 14-3-3 , , , , , , and  (2 ng/lane) were included. Staining Kit (Catalog # CTS008; brown) and counterstained with hematoxylin (blue). 14-3-3 was detected at approximately 28 kDa (as indicated).

www.RnDSystems.com/go/Apoptosis 69 NEURODEGENERATIVE DISEASES

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Caspases & Regulators, continued SMAC/Diablo HH (IHC, WB) H

cIAP-2/HIAP-1 HH (IHC, WB) M (IHC, WB) Survivin HH (FC, IHC, WB) H

cIAP H (WB) M (WB) TopBP1 H (IHC)

FLIP HH (WB) M (WB) XIAP HH (B/N, IHC, WB) M (IHC, WB) R (IHC, WB) H

Livin HH (WB) H Mitochondrial Proteins

NAIP H (IHC, WB) AIF H (IHC, WB) M (IHC, WB) R (IHC, WB)

NALP1 H (WB) COX4 H (IHC, WB) M (IHC, WB)

NALP2 H (WB) COX4-I1 H (IHC, WB) M (IHC, WB)

NALP3 H (IHC, WB) COX4-I2 H (WB) M (WB)

Pynod H (WB) M (WB) R (WB) Cytochrome c HH (IHC, IP, WB) M (IP, WB) R (IP, WB) E (IHC, IP) H M R

Survivin HH (FC, IHC, WB) H HTRA2/Omi HH (IHC, WB) M (IHC, WB) R (IHC, WB)

XIAP HH (B/N, IHC, WB) M (IHC, WB) R (IHC, WB) H PDHX H (WB) M (WB) R (WB)

Granzymes SMAC/Diablo HH (IHC, WB) H

Granzyme A HH (FC, IHC, IP, WB) UCP1 H (WB) M (WB)

Granzyme BH MH (FC, IHC, WB) M (B/N, ELISA, IHC, IP, WB) M UCP2 H (WB) M (WB)

Granzyme D MM (IHC, IP, WB) NFB Pathway

Granzyme G MM (IP, WB) A20/TNFAIP3 H

Granzyme H HH (IHC, IP, WB) BCR H (WB)

Heat Shock Proteins CARD11/CARMA1 H (WB) M (WB) R (WB)

Clusterin H M H (ELISA, IHC, IP, WB) M (ELISA, IHC, IP, WB) H M CARD9 H (WB)

AlphaA Crystallin/ H (WB) M (WB) FAT10 H CRYAA Gliotoxin Ms AlphaB Crystallin/ H (WB) M (WB) R (WB) CRYAB IkB- H (WB) M (WB) H M R

GRP75/HSPA9B H (IHC, WB) M (IHC, WB) R (IHC, WB) IkB- H (WB) M (WB) R (WB)

GRP78/HSPA5 H (WB) M (WB) R (WB) IkB- H (WB) M (WB)

HO-1/HMOX1/HSP32 H (WB) M (WB) R (WB) H IKK H (IHC, WB) M (IHC, WB) R (IHC, WB)

HSP10/EPF H (WB) M (WB) IKK H (WB) M (WB)

HSP20/HSPB6 H (WB) M (WB) R (WB) IKK H (IHC, WB) M (IHC, WB) R (IHC, WB)

HSP27 HH (WB) M (WB) R (WB) H M R IKK H (IHC, WB) M (IHC, WB) R (IHC, WB)

HSP40/DNAJB1 H (WB) M (WB) R (WB) LRRC4 HH (IHC, WB) M (WB)

HSP60 H (IHC, WB) M (IHC, WB) R (IHC, WB) H MALT1 H (WB)

HSP70/HSPA1A H (IHC, WB) M (IHC, WB) R (WB) H M R MEKK3 H (WB)

HSP90 H (WB) M (WB) R (WB) NIK/MAP3K14 H (IHC)

HSPA2 H (WB) M (WB) R (WB) PIAS3 H (WB)

HSPA8/HSC71 H (WB) M (WB) R (WB) Pin1 HH (IHC, IP, WB) M (IHC, IP, WB) H M

HSPB8 H (WB) M (WB) R (WB) RelA/NFB p65 H (ChIP, FC, WB) M (ChIP, FC, WB) H

HSPH1 H (IHC, WB) M (IHC, WB) R (IHC, WB) RelB H (IHC, WB)

ORP150/HSP12A H (WB) c-Rel H (ChIP, IHC, WB) M (ChIP, IHC, WB) H Inhibitors of Apoptosis (IAPs) & Regulators RIP1 H (WB) M (WB) R (WB)

cIAP-1/HIAP-2 HH (IHC, WB) M (WB) SENP1 H

cIAP-2/HIAP-1 HH (IHC, WB) M (IHC, WB) SENP2 H

cIAP H (WB) M (WB) STING/TMEM173 H (FC, WB)

CIDEA H (WB) SUMO1-Specifi c H Peptidase 1/SENP1 CIDEC H (IHC, WB) SUMO1-Specifi c H HTRA2/Omi HH (IHC, WB) M (IHC, WB) R (IHC, WB) Peptidase 2/SENP2

Livin HH (WB) H SUMO1 HH (WB) M (WB) Ms

NAIP H (IHC, WB) SUMO1/2/3 H

PAWR H (IHC) SUMO2/3/4 H (WB) M (WB)

70 For research use only. Not for use in diagnostic procedures. NEURODEGENERATIVE DISEASES

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

SUMO2 HH (WB) M (WB) Ms COX4-I1 H (IHC, WB) M (IHC, WB)

Poly-SUMO2 H COX4-I2 H (WB) M (WB)

Di-SUMO2 H Glutaredoxin 1/ H (WB) M (WB) GLRX1 SUMO3 HH (IHC, WB) M (WB) Ms Glutathione H (IHC, WB) M (IHC, WB) R (IHC, WB) Poly-SUMO3 H Peroxidase 1/GPX1

Di-SUMO3 H Glutathione H (WB) M (WB) R (WB) Peroxidase 2/GPX2 SUMO4 H (WB) M (WB) Glutathione H (IHC, WB) M (IHC, WB) R (IHC, WB) TAK1 H (WB) Peroxidase 3/GPX3 UBE2N/Ubc13 H Glutathione H (WB) M (WB) R (WB) UBE2N (Ubc13)/ H Peroxidase 4/GPX4 Uev1a Complex Glyoxalase I HH (WB) M (WB) R (WB) Ubiquitin H Pz Pl Y Ms H (IHC, WB) Ms Glyoxalase II HH (WB) M (WB) R (WB) Ubiquitin/ H (IHC, WB) Ubiquitin+1 Haptoglobin M (WB) HAX-1 H (WB) Ubiquitin+1 HH (ELISA, IHC, WB) H Di-Ubiquitin/Ub2 H Ms HO-2/HMOX2 H (WB) M (WB) R (WB) Myeloperoxidase/ H M H (IHC, WB) M (IHC, WB) H Tri-Ubiquitin/Ub3 H MPO Tetra-Ubiquitin/Ub4 H Nitric Oxide Ms Penta-Ubiquitin/Ub5 H Nitrotyrosine Ms (IHC, WB) Hexa-Ubiquitin/Ub6 H eNOS H (IHC, IP, WB) H Octa-Ubiquitin/Ub8 H iNOS H (IHC, WB) H Poly-Ubiquitin H nNOS H (IHC, WB) Uev1a (Mms2)/ H UBE2V1 Nucleoredoxin H (WB) Z-Leu-Leu-Leu-CHO Ms Park7/DJ-1 H (IHC, WB) M (WB) H (MG-132) Peroxiredoxin 1 H (WB) M (WB) R (WB)

Oxidative Stress Peroxiredoxin 2 H (WB) M (WB) R (WB)

4-Hydroxynonenal Ms (WB) Peroxiredoxin 3 H (WB) M (WB)

Carboxymethyl Ms (WB) Peroxiredoxin 4 H (WB) M (WB) R (WB) Lysine Peroxiredoxin 5 H (WB) M (WB) R (WB) Catalase H (WB) M (WB) R (WB) Peroxiredoxin 6 H (WB) M (WB) R (WB) COX4 H (IHC, WB) M (IHC, WB)

* UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat E Equine F Feline Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot Y 5 an t

A. B. r m u ea

kDa H H 100 kDa SH-SY 113 94 93 80 65 65 Pro-Caspase-10 60 39 40

23 SMAC/Diablo 40 Number Cell Relative 19 20

0 6 24 100 101 102 103 104 Bcl-6

Detection of Human SMAC/Diablo and Pro-Caspase-10 by Western Blot. Western XIAP in Human Cortex. X-chromosome linked inhibitor of apoptosis (XIAP) was Detection of Bcl-6 by Flow Cytometry. The Raji human Burkitt’s lymphoma cell blots show lysates of the SH-SY5Y human neuroblastoma cell line and human heart detected in immersion-fi xed paraffi n-embedded sections of human cortex using a line was stained with a Rat Human/Mouse Bcl-6 Monoclonal Antibody (Catalog tissue. A. The PVDF membrane was probed with a Goat Anti-Human SMAC/Diablo Mouse Anti-Human XIAP Monoclonal Antibody (Catalog # MAB8221). The tissue was # MAB5046, fi lled histogram) or a Rat IgG2B Isotype Control (Catalog # MAB0061, Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF7891) followed by a HRP- stained with the Anti-Mouse HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS002; open histogram), followed by a PE-conjugated Anti-Rat IgG Secondary Antibody conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # HAF109). B. The PVDF brown) and counterstained with hematoxylin (blue). Specifi c staining was localized (Catalog # F0105B). To facilitate intracellular staining, the cells were fi xed with membrane was probed with a Goat Anti-Human Pro-Caspase-10 Antigen Affi nity- to the cytoplasm of cortical neurons. paraformaldehyde and permeabilized with saponin. purifi ed Polyclonal Antibody (Catalog # AF6864) followed by a HRP-conjugated Rabbit Anti-Goat IgG Secondary Antibody (Catalog # HAF017). SMAC/Diablo and Pro-Caspase-10 were detected at approximately 23 kDa and 60 kDa, respectively (as indicated). www.RnDSystems.com/go/Apoptosis 71 NEURODEGENERATIVE DISEASES

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Oxidative Stress, continued VRK1 H (WB)

PON1 H (WB) M (WB) H WISP-1/CCN4 H M H (ELISA, IHC, WB) M (IHC, WB) H

PON2 H (WB) H TNF Superfamily

PON3 H (WB) M (WB) R (WB) H 4-1BB/TNFRSF9/ H M H (ELISA, FA, FC, IHC, WB) M (B/N, ELISA, FA, FC, WB) H M CD137 Renalase H (WB) 4-1BB Ligand/ H M H (FC, WB) M (FC, WB) SOD (Superoxide Ms TNFSF9 Dismutase) APRIL/TNFSF13 HH (ELISA, FC, WB) M (FC) H SOD1/Cu-Zn SOD H (IHC, WB) M (WB) R (WB) BAFF/BLyS/ H M H (B/N, FC, IHC, WB) M (B/N, FC, IHC, WB) H M SOD2/Mn-SOD H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R TNFSF13B

SOD3/EC-SOD H (WB) M (WB) R (WB) BAFF R/TNFRSF13C H M H (B/N, FC, WB) M (B/N, ELISA, FC, WB) M

Thioredoxin-1 HH (IHC, WB) BCMA/TNFRSF17 H M H (B/N, ELISA, FC, WB) M (B/N, ELISA, FC, WB) H

Thioredoxin-2 H (WB) M (WB) R (WB) CD27/TNFRSF7 H M H (B/N, FC, IHC, WB) M (ELISA, FC, WB) M

Thioredoxin-80 H CD27 Ligand/TNFSF7 MH (FC, IHC, WB) M (B/N, ELISA, FC, WB) M

Thioredoxin-like 5/ H (WB) M (WB) R (WB) CD30/TNFRSF8 H M H (FA, FC, WB) M (ELISA, FA, IHC, WB) M TRP14 CD30 Ligand/TNFSF8 H M H (B/N, FC, WB) M (B/N, ELISA, FC, WB) M Thioredoxin H (IHC, WB) M (IHC, WB) Reductase 2/TRXR2 CD40/TNFRSF5 H M H (B/N, FA, FC, IHC, WB) M (ELISA, FA, FC, IF, IHC, IP, WB) M

TXNDC5 H (WB) CD40 Ligand/TNFSF5 H M H (B/N, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M p53 Pathway CRMB V

53BP1 H (WB) M (WB) R (WB) Daxx H (WB)

Ac-Leu-Leu-Nle-CHO Ms DcR3/TNFRSF6B HH (B/N, ELISA, IHC, WB) H (MG-101) DcTRAIL R1/ M (IHC, WB) AP-2 H (IHC, WB) TNFRSF23

ATM H (IHC, WB) M (WB) R (WB) H DcTRAIL R2/ MM (WB) TNFRSF22 ATR H (WB) DR3/TNFRSF25 H M H (FC, WB) M (IHC, WB) CBP H (IHC, WB) M (IHC, WB) R (IHC, WB) DR6/TNFRSF21 H M H (ELISA, WB) H Clathrin Heavy Chain H (WB) 1/CHC17 EDA/Ectodysplasin H (ELISA) H

Clathrin Heavy Chain H (WB) EDA-A1/ H M 2/CHC22 Ectodysplasin A1

HLI 373 Ms EDA-A2/ HH (B/N, WB) Ectodysplasin A2 HR6A/UBE2A H EDAR H M H (B/N, WB) M (B/N, ELISA, WB) M HR6B/UBE2B H Fas/TNFRSF6/CD95 H M R F H (ELISA, FC, IHC, WB) M (ELISA, FC, IHC, WB) H M IFITM2 H (WB) R (FC, IHC, WB) F (FC, IHC, WB)

IFITM2/IFITM3 H (WB) Fas Ligand/TNFSF6 H M R H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, WB) H M R (IHC, WB) ING1 H (IHC, WB) M (WB) GITR/TNFRSF18 H M H (B/N, ELISA, FC, IHC, WB) M (ELISA, FC, WB) H M LKB1 H (WB) GITR Ligand/ H M H (B/N, ELISA, FC, WB) M (B/N, ELISA, WB) H M MDM2/HDM2 H (IHC, IP, WB) M (IHC, WB) R (IHC, WB) H TNFSF18 NPM1 H (WB) M (WB) HVEM/TNFRSF14 H M H (ELISA, FC, IHC, WB) M (WB) H NSC 146109 Ms Hydrochloride LIGHT/TNFSF14 H M H (B/N, ELISA, FC, WB) M (FC, WB) H NSC 66811 Ms Lymphotoxin H M H (B/N) Lymphotoxin / H (WB) p53 HH (ChIP, FC, IHC, IP, WB) M (ChIP, IP, WB) R (ChIP, IP, WB) H M TNFSF3 p53R2 H (WB) Lymphotoxin βR/ H M H (B/N, FC, WB) M (FC, IHC, WB) p300 H (ChIP, IHC, WB) H TNFRSF3 PYR 41 Ms NGF R/TNFRSF16 H M H (FC, IHC, WB) M (IHC, WB)

RTVP-1 M (WB) Osteoprotegerin/ H M H (B/N, ELISA, IHC, WB) M (B/N, ELISA, IHC, WB) H M TNFRSF11B Thalidomide Ms OX40/TNFRSF4 H M H (FC, WB) M (FA, FC, WB) TRF2 H (WB) OX40 Ligand/TNFSF4 H M H (B/N, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) M USP7 H RANK/TNFRSF11A H M H (ELISA, FA, FC, IHC, WB) M (FA, IHC, WB) H USP8 H

72 For research use only. Not for use in diagnostic procedures. NEURODEGENERATIVE DISEASES

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

RELT/TNFRSF19L HH (FC, WB) Additional Apoptosis Molecules

TACI/TNFRSF13B H M H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M AATK H (FC, WB)

TANK H (WB) M (WB) Annexin V HH (WB) Ms

TL1A/TNFSF15 H M H (WB) M (WB) Apolipoprotein H/ H M H (WB) ApoH TNF- H M R P B Ca CR H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M R P B Ca E F Rb RM R (B/N, ELISA, IHC, WB) B (ELISA, IHC, WB) CR E F Pr Rb BAT3 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ca (B/N, ELISA, IHC, WB) CR (B/N, WB) E (B/N, IHC, WB) RM GP (B/N, ELISA, IHC, WB) Complexin-2 H (WB) M (WB) R (WB)

TNF-/TNFSF1 H M H (B/N, ELISA, FC, IHC, WB) M (IHC, WB) H CRF21 H (WB)

TNFRH3/TNFRSF26 M (FC, WB) CSE1L/CAS H (WB) M (WB) R (WB)

TNF RI/TNFRSF1A H M Ca H (B/N, ELISA, FA, FC, IHC, WB) M (B/N, ELISA, FA, FC, IHC, IP, H M CTGF/CCN2 H (IHC, WB) WB) Cyr61/CCN1 HH (WB) M (IHC, WB) TNF RII/TNFRSF1B H M H (B/N, ELISA, FC, IHC, WB) M (ELISA, FC, IHC, IP, WB) H M DIDO1 H (WB) TRADD H (FC, IHC, WB) NMNAT-1 HH (IHC, IP, WB) TRAF-1 H (WB) NMNAT-2 H TRAF-2 H (WB) M (WB) R (WB) OAS2 H (IHC, IP) TRAF-3 H (WB) M (WB) R (WB) PARP H (IP, WB) M (IP, WB) Ms (IHC, WB) Ms TRAF-4 H (WB) PBR H M R H (IHC, WB) M (IHC, WB) R (IHC, WB) TRAF-6 H (WB) PDCD4 H (IHC) TRAIL/TNFSF10 H M H (B/N, ELISA, FC, IHC, WB) M (ELISA, IHC, WB) H M Porimin H (FC, WB) TRAIL R1/TNFRSF10A HH (B/N, FC, IHC, WB) PSR M (WB) TRAIL R2/TNFRSF10B H M H (FA, FC, WB) M (B/N, FC, IHC, WB) RASSF2 H (WB) M (WB) TRAIL R3/TNFRSF10C HH (B/N, ELISA, FC, IHC, WB) H Stathmin/STMN1 H (WB) M (WB) R (WB) TRAIL R4/TNFRSF10D HH (B/N, ELISA, FC, IHC, WB) H Thymosin β4 H (IHC, WB) TRANCE/TNFSF11/ H M H (B/N, IHC, WB) M (B/N, ELISA, IHC, WB) M RANK L Thymosin β10 H (WB) Transglutaminase 2/ H M H (WB) M (WB) R (WB) TROY/TNFRSF19 H M H (WB) M (ELISA, IHC, WB) M TGM2 TWEAK/TNFSF12 H M H (B/N, ELISA, IHC, WB) M (B/N, ELISA, WB) H M Transglutaminase 7/ HH (WB) TWEAK R/TNFRSF12 H M H (B/N, IHC, WB) M (ELISA, FC, IHC, WB) M TGM7

XEDAR HH (B/N, ELISA, WB) H

* UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot 0 00 0 1 3 1 30 1 300

A 600 2

A.ro- B. NIH-3T3 iNOS eu

kDa N kDa –+Heat 500 113 GAPDH 93 400 65 94 HSP70 65 300 40 200 39

24 RFUs) iNOS (Normalized Total Bad 100 23 18 0 0 1 3 10 30 100 300 IFN-J + IL-1E + TNF-D (ng/mL)

Detection of Mouse Bad and HSP70 by Western Blot. A. Western blot shows NAIP in Human Cerebellum. Neuronal apoptosis inhibitory protein (NAIP) was Detection of iNOS by Cell-Based ELISA and Western Blot. HT-29 human colon lysates of the Neuro-2A mouse neuroblastoma cell line. The PVDF membrane was detected in immersion-fi xed paraffi n-embedded sections of human cerebellum adenocarcinoma cells were cultured in 96-well microplates and treated with the probed with a Mouse Anti-Human Bad Monoclonal Antibody (Catalog # MAB6405) using a Mouse Anti-Human NAIP Monoclonal Antibody (Catalog # MAB829). Before indicated amounts of Recombinant Human IFN- (Catalog # 285-IF), Recombinant followed by a HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody (Catalog # incubation with the primary antibody, the tissue was subjected to heat-induced Human IL-1 (Catalog # 201-LB), and Recombinant Human TNF- (Catalog # 210- HAF007). B. Western blot shows lysates of the NIH-3T3 mouse embryonic fi broblast epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). The TA) for 24 hours. After fi xation of cells in the wells, iNOS expression was determined cell line untreated (–) or treated (+) with a 42 °C heat shock for 30 minutes with a 3 tissue was stained using the Anti-Mouse HRP-DAB Cell & Tissue Staining Kit (Catalog using the Human Total iNOS Cell-Based ELISA (Catalog # KCB9502, bar chart). Values hour recovery. The PVDF membrane was probed with a Mouse Anti-Human/Mouse # CTS002; brown) and counterstained with hematoxylin (blue). Specifi c staining was represent mean + range of duplicate determinations. Analysis of iNOS and GAPDH HSP70 Monoclonal Antibody (Catalog # MAB1663), followed by a HRP-conjugated localized to Purkinje neurons in the cerebellum. (the normalization housekeeping protein) by Western blot, using the antibodies Goat Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). Bad and HSP70 were supplied in this kit, is also shown (inset). detected at approximately 23 kDa and 70 kDa, respectively (as indicated). www.RnDSystems.com/go/Apoptosis 73 SIGNAL TRANSDUCTION

Signal Transduction An important aspect of any biological study is elucidating the signal transduction mechanism that underlies the key fi nding. Revealing the changes in intracellular activity and/or gene expression that govern the actions of neural cells often presents novel molecular targets for further experimentation and pharmacological manipulation. R&D Systems off ers a wide variety of high quality tools to investigate signal transduction cascades in neurons, glia, and oligodendrocytes. These include products to study changes in function, expression, and post-translational modifi cation. Our catalog includes tools to elucidate neural actions that are dependent on receptors, second messenger molecules, adaptor proteins, intracellular enzymes, phosphorylation state, Ca2+-binding proteins, transcription factors, and translational regulators. For a wide selection of chemically-based small compounds & peptides please visit: R&D Systems Products for Signal Transduction www.Tocris.com RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Adaptor Proteins Gab2 H (WB)

14-3-3 H (WB) M (WB) R (WB) B (WB) Ca (WB) Ch (WB) Pr (WB) GAPDH/G3PDH H (IHC, IP, WB) M (IHC, WB) R (IHC, WB) X (WB) Z (WB) GAPDH-2 H (IHC, WB) 14-3-3 H (WB) M (WB) R (WB) GL H (WB) M (WB) R (WB) 14-3-3 H (WB) M (WB) R (WB) GRAP2 H (WB) M (WB) 14-3-3 H (WB) M (WB) R (WB) GRB2 H (IHC, WB) M (IHC, WB) R (IHC, WB) 14-3-3 H (WB) M (WB) R (WB) GRB7 H (WB) M (WB) R (WB) 14-3-3 H (WB) M (WB) R (WB) IRS1 H (IHC, WB) M (WB) R (WB) 14-3-3 H (WB) M (WB) R (WB) IRS2 H (WB) 14-3-3 HH (WB) M (WB) KPNA1 H (WB) APBA2 H (WB) M (WB) R (WB) KPNA2 H (IHC, WB) APBA3 H (WB) LAT H (FC, WB) APPL H (WB) LNK/SH2B3 H (WB) BAP31 H (WB) Mena H (WB) BCAP H (WB) M (WB) MIG2 H (WB) BLNK H (WB) MyD88 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H BRDG1 H (WB) NCK1 H (WB) CARD9 H (WB) NTAL H (FC, WB) CBL H (WB) M (WB) Numb H (IHC, WB) H M R CD2AP H (WB) M (WB) R (WB) p130Cas H (WB) M (WB) R (WB) CIDEC H (IHC, WB) PAG1 H (WB) CRADD H (WB) M (WB) R (WB) Paxillin H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R Crk H (WB) M (WB) PDHX H (WB) M (WB) R (WB) CrkL H (WB) M (WB) R (WB) PINCH1 H (WB) M (WB) R (WB) DAPP1 H (WB) M (WB) Ras-GAP H (WB) M (WB) R (WB) Daxx H (WB) RIBP/SH2D2A M (WB) R (WB) DFF40/CAD H (WB) SAM68 H (WB) M (WB) DFF45/ICAD H (WB) SH2B1 H (WB) DOCK1 H (WB) R (WB) SH2D2A H (WB) DOCK2 H (WB) SHANK2 H (IHC) DOK3 H (WB) SHB H (IHC) DOK4 H (IHC, WB) SIT1 H (WB) DOK7 H (WB) M (WB) R (WB) SOCS-5 H (IHC, WB) FADD H (IHC, WB) SOCS-6 H (WB) FLIP HH (WB) M (WB) SOCS-7/Nck/NAP4 H (WB) FRS2 H (FC, IHC, WB) M (IHC, WB) R (IHC, WB) H M R STUB1/CHIP H (WB) GAB1 H (WB) SWAP70 H (WB) M (WB)

74 For research use only. Not for use in diagnostic procedures. SIGNAL TRANSDUCTION

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

TANK H (WB) M (WB) AMPK1 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R

TAPP1 H (WB) AMPK2 H (IHC, WB) M (IHC, WB) R (IHC, WB)

Tollip H (WB) R (WB) AMPK1 H (IHC, WB) M (IHC, WB) R (IHC, WB)

TRADD H (FC, IHC, WB) AMPK2 H (WB) M (WB)

TRAF-1 H (WB) A-Raf H (WB)

TRAF-2 H (WB) M (WB) R (WB) ARK5 H (WB) M (WB) R (WB)

TRAF-3 H (WB) M (WB) R (WB) ASK1 HH (IHC, WB)

TRAF-4 H (WB) ATM H (IHC, WB) M (WB) R (WB) H

TRAF-6 H (WB) Aurora A H (WB)

TRAM/TICAM2 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) Aurora B H (WB) M (WB)

TRIF/TICAM1 H (IHC, WB) Bad H (IHC, WB) M (WB) H

UBASH3B/STS1 H (IHC, WB) M (IHC, WB) R (IHC, WB) -Catenin H (ChIP, FC, IHC, WB) M (ChIP, FC, IHC, WB) H R (ChIP, FC, IHC, WB) X (WB) Vav-1 H (WB) M (WB) BCR H (WB) Circadian Rhythm Molecules Blk HH (WB) M (WB) R (WB) CLOCK H (WB) BMX H (IHC, WB) CRY1 H (IHC, WB) M (IHC, WB) Brk H (IHC, WB) ELF3 H (IHC, WB) M (IHC, WB) BTK H (IHC, WB) PER1 H (IHC) CaM Kinase II H (WB) M (WB) R (WB) X (WB) Intracellular Kinases CaM Kinase II H (IHC) 4EBP1 H (WB) M (WB) CaM Kinase II H (IHC, WB) A20/TNFAIP3 H CaMKK H (WB) R (WB) AAK1 H (WB) CARD11/CARMA1 H (WB) M (WB) R (WB) AATK H (FC, WB) Casein Kinase 1 H (WB) M (WB) R (WB) Akt H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R Casein Kinase 1 H (WB) M (WB) R (WB) Akt1 HH (FC, IHC, WB) M (WB) R (WB) H M R Casein Kinase 1δ H (WB) M (WB) R (WB) Akt2 H (FC, IHC, WB) M (IHC, WB) R (IHC, WB) H Casein Kinase 1 H (WB) M (WB) R (WB) Akt3 H (FC, WB) Casein Kinase 2 H (WB) AMPK1/2 H (WB) Chk1 HH (IP, WB) M (WB) R (WB) H M R * UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot ain ain s u Br Br ain m e an m s a l Br u u m ll u at e x tha R H Mo b

kDa te e ain

80 r or Br Ce C Hypo

ain e e 94 e an s s 70 s Br u u u m u kDa at Mo Mo 65 60 122 R H Mo 50 94 40 65 CK1J 39 GAPDH 30 39 Relative Cell Number Cell Relative 20 10 0 23 100 101 102 103 104 23 Akt1 19

Detection of GAPDH by Western Blot. Western blot shows lysates of mouse, Detection of Akt1 by Flow Cytometry. The MCF-7 human breast cancer cell line Detection of Casein Kinase 1 by Western Blot. Western blot shows lysates of human, and rat brain tissue. The PVDF membrane was probed with a Goat Anti- was stained with a Mouse Anti-Human Akt1 Monoclonal Antibody (Catalog # human, mouse, and rat brain tissue. The PVDF membrane was probed with a Sheep

Human/Mouse/Rat GAPDH/G3PDH Antigen Affi nity-purifi ed Polyclonal Antibody MAB17751, fi lled histogram) or a Mouse IgG1 Isotype Control Antibody (Catalog Anti-Human/Mouse/Rat Casein Kinase 1 Antigen Affi nity-purifi ed Polyclonal (Catalog # AF5718) followed by a HRP-conjugated Rabbit Anti-Goat IgG Secondary # MAB002, open histogram), followed by a PE-conjugated Goat Anti-Mouse IgG Antibody (Catalog # AF3186) followed by a HRP-conjugated Donkey Anti-Sheep IgG Antibody (Catalog # HAF017). GAPDH/G3PDH was detected at approximately 38 kDa Secondary Antibody (Catalog # F0102B). To facilitate intracellular staining, cells Secondary Antibody (Catalog # HAF016). Casein Kinase 1 isoforms were detected (as indicated). were fi xed with paraformaldehyde and permeabilized with saponin. at approximately 45 to 51 kDa (as indicated).

www.RnDSystems.com/go/SignalTransduction 75 SIGNAL TRANSDUCTION

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Intracellular Kinases, continued IkB- H (WB) M (WB) R (WB)

Chk2 HH (IHC, WB) M (IHC, WB) R (WB) H M R IkB- H (WB) M (WB)

DAPK3/ZIPK H (WB) IKK H (IHC, WB) M (IHC, WB) R (IHC, WB)

DGK- H (WB) IKK H (WB) M (WB)

DGK- H (WB) IKK H (IHC, WB) M (IHC, WB) R (IHC, WB)

DGK- H (WB) M (WB) R (WB) IKK H (IHC, WB) M (IHC, WB) R (IHC, WB)

DGK- H (IHC, WB) M (IHC, WB) R (WB) ILK H (WB) M (WB) R (WB)

DGK- H (WB) M (WB) IRAK2 H (WB)

DGK- H (WB) M (WB) R (WB) IRAK1 H (WB)

DGK- H (WB) IRAK3 H (WB)

DISC1 H (IHC, WB) IRAK4 H (IHC, WB)

DMPK H (WB) ITK H

DRAK1 H (WB) Jak1 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB)

DRAK2 H (WB) M (WB) R (WB) Jak2 M (WB) R (WB)

DYRK1A H (IHC, WB) R (IHC, WB) Jak3 H (FC, WB)

DYRK2 H (IHC, WB) M (WB) R (IHC, WB) JNK H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R

DYRK3 H (WB) M (WB) R (WB) JNK1/JNK2 H (IHC, WB) M (IHC, WB) R (IHC, WB)

ERK1/ERK2 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R JNK1 MH (IHC, WB) M (IHC, WB) R (IHC, WB)

ERK1 HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R JNK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R

ERK2 HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R Lck HH (FC, IHC, WB)

ERK3 H (IHC, WB) LKB1 H (WB)

ERK4 H (IHC, WB) M (IHC, WB) R (IHC, WB) LMTK2 H (WB)

ERK5/BMK1 H (IHC, WB) M (IHC, WB) LRRC4 HH (IHC, WB) M (WB)

FAK HH (IHC, WB) M (IHC, WB) R (IHC, WB) H M R LRRK2 H (IHC)

FAT10 H LTK H (FC, IHC, WB)

Fes H (IHC, WB) Lyn H (IHC, WB) M (IHC, WB) R (IHC, WB) H

Fgr H (IHC, WB) M (WB) R (IHC, WB) Lyn B H

FoxD3 H (IHC, WB) M (WB) MALT1 H (WB)

FoxJ1 H (FC, IHC, WB) MAP Kinase Array Kit

FoxP3 H (ChIP, FC, IHC, WB) M (FC) R (FC) H MAP3K8/Tpl2/COT HH (WB)

Frk H (IHC, WB) M (IHC, WB) R (IHC, WB) MAPKAPK2 H

Fyn H (IHC, WB) M (WB) R (WB) MARCKS H (WB) M (WB) R (WB) B (WB) Ch (WB) X (WB) Z (WB)

Girdin H (WB) MDM2/HDM2 H (IHC, IP, WB) M (IHC, WB) R (IHC, WB) H

Gliotoxin Ms MEK1/MEK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R

Glut4 R (AP, IHC, IP, WB) MEK1 H (IHC, WB) M (IHC, WB) R (IHC, WB) B (WB) Ca (WB) Ch (WB) Pr (WB) X (WB) GRK1 H (WB) M (WB) R (WB) MEK2 HH (IHC, WB) M (IHC, WB) R (IHC, WB) GRK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) MEKK3 H (WB) GRK3 H (WB) MELK H (IHC, WB) GRK5 H (WB) M (WB) R (WB) MKK3/MKK6 H (WB) M (WB) R (WB) GRK7 H (WB) M (WB) R (WB) MKK3 H (IHC, WB) M (IHC, WB) R (IHC, WB) H GSK-3/ H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R MKK4 H (IHC, WB) M (IHC, WB) R (IHC, WB) GSK-3 H (IHC, WB) M (IHC, WB) R (IHC, WB) H MKK6 H (IHC, WB) M (IHC, WB) R (IHC, WB) H GSK-3 HH (FC, IHC, WB) M (FC, WB) R (FC, WB) H M R MKK7 H (IHC, WB) Hck H (WB) M (WB) MLK2 H (WB) HGK H (WB) MLK3 H (WB) HIPK1 H (WB) MLK4 H (IHC, WB) M (IHC, WB) R (IHC, WB) IkB- H (WB) M (WB) H M R MSK1/MSK2 H (WB)

76 For research use only. Not for use in diagnostic procedures. SIGNAL TRANSDUCTION

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

MSK1 H (IHC, WB) M (IHC, WB) PKC H (WB) M (WB) R (WB)

MSK2 H (IHC, WB) M (IHC, WB) PKC1 H (IHC, WB) R (IHC, WB)

MST1/STK4 H (WB) PKC2 H (WB) M (WB)

MST2/STK3 H (WB) M (WB) R (WB) PKC H (WB) M (WB) R (WB)

NEK2 H PKC H (IHC, WB) M (IHC, WB) R (IHC, WB)

NFB1 H (ChIP, WB) M (ChIP, WB) H PKC HH (WB)

NFB2 H (ChIP, IHC, WB) M (WB) H PKC/ H (WB) M (WB)

NIK/MAP3K14 H (IHC) PKC// H (IHC, WB) M (IHC, WB) R (IHC, WB)

NM23-H1 H (IHC, WB) M (IHC, WB) R (IHC, WB) PKC H (WB)

NM23-H1/H2 H (IHC, WB) M (IHC, WB) R (IHC, WB) PKC H (FC, IHC, WB) M (WB)

NM23-H2 H (IHC, WB) M (IHC, WB) R (IHC, WB) PKC H (WB)

p27/Kip1 H (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H PKN1 H (WB) M (WB)

p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M PKN2 H (WB) M (WB) R (WB)

p38 HH (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M R PKR H (IHC, IP, WB)

p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M PLK1 HH (WB)

p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M R PLK3 H (WB)

p38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Ms (IP) H M PLKK H (WB) M (WB) R (WB) X (WB) Z (WB)

p70 S6 Kinase HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R PP2A H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R

p70 S6 Kinase  H (WB) PPP2R4 H (WB) M (WB) R (WB)

p55  H (WB) M (WB) PRAS40 H (WB)

PAK H (WB) M (WB) R (WB) Pr (WB) PTEN HH (FC, IHC, WB) M (FC, IHC, WB) R (FC, IHC, WB) H M R

PAK2 H (WB) PYK2/FAK2 HH (WB)

PAK3 H (IHC, WB) M (IHC, WB) Raf-1 HH (IHC, WB) M (IHC, WB) R (IHC, WB) B (WB) Ca (WB) Ch (WB) Pr (WB) PAK4 H (WB) M (WB) R (WB) B-Raf H (WB) M (WB) R (WB) PAK6 H (WB) M (WB) R (WB) RelA/NFB p65 H (ChIP, FC, WB) M (ChIP, FC, WB) H PAK7 H (IHC, WB) R (IHC, WB) RelB H (IHC, WB) PCK1 H (WB) M (WB) R (WB) c-Rel H (ChIP, IHC, WB) M (ChIP, IHC, WB) H PDK-1 HH (WB) M (WB) R (WB) RIP1 H (WB) M (WB) R (WB) PI 3-Kinase p55 H (WB) M (WB) R (WB) RLK/TXK H (WB) PI 3-Kinase p85 H (WB) M (WB) R (WB) ROCK1 HH (WB) M (WB) R (WB) PI 3-Kinase p110 H (WB) ROCK2 H (WB) M (WB) R (WB) PI 3-Kinase p110 H (WB) RSK H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R PI 3-Kinase p110 H (WB) RSK1/RSK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) PI 3-Kinase p85 H (WB) RSK1 HH (IHC, WB) M (IHC, WB) R (IHC, WB) H PIAS3 H (WB) RSK2 H (IHC, WB) M (IHC, WB) R (IHC, WB) PIK3R4 H (WB) RSK3 H (IHC, WB) M (IHC, WB) PIK3R5 H (WB) RSK4 H (WB) PIM1 H SUMO1-Specifi c H PIM2 H (WB) M (WB) R (WB) Peptidase 1/SENP1 Pin1 HH (IHC, IP, WB) M (IHC, IP, WB) H M SUMO1-Specifi c H Peptidase 2/SENP2 PINK1 H (IHC) SGK HH (WB) PKA C H (WB) M (WB) R (WB) SHIP2 H (IHC, WB) M (IHC, WB) R (IHC, WB) PKA C HH (WB) M (WB) R (WB) SNF1LK2 H (IHC, WB) PKA C/ H (WB) M (WB) R (WB) Sphingosine Kinase H M H (IP, WB) PKA C HH (WB) M (WB) R (WB) 1/SPHK1 PKA RI H (WB) M (WB) R (WB) * UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot

www.RnDSystems.com/go/SignalTransduction 77 SIGNAL TRANSDUCTION

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Intracellular Kinases, continued DAX1/NR0B1 H (WB)

Src H V H (IHC, WB) M (IHC, WB) R (IHC, WB) H EAR2/NR2F6 H (IHC, IP, WB)

SRPK2 H (WB) ER/NR3A1 H (IHC, IP, WB) M (WB) R (WB) H

STING/TMEM173 H (FC, WB) ER/NR3A2 H (IP, WB)

SUMO1 HH (WB) M (WB) H ERR/NR3B1 H (IHC, IP, WB)

SUMO1/2/3 H ERR/NR3B2 H (IHC, IP, WB)

SUMO2/3/4 H (WB) M (WB) ERR/NR3B3 H (IHC, IP, WB)

SUMO2 HH (WB) M (WB) H FXR/NR1H4 H (IHC, IP, WB)

Poly-SUMO2 H GCNF/NR6A1 H (IHC, IP, WB)

Di-SUMO2 H GR/NR3C1 H (IHC, IP, WB)

SUMO3 HH (IHC, WB) M (WB) H HNF-4α/NR2A1 H (EMSA, IHC, IP, WB)

Poly-SUMO3 H HNF-4/NR2A2 H (IHC, WB)

Di-SUMO3 H LRH-1/NR5A2 H (IP, WB)

SUMO4 H (WB) M (WB) LXRα/NR1H3 H (EMSA, IHC, IP, WB)

SYK HH (WB) LXR/NR1H2 H (IP, WB)

TAK1 H (WB) Mineralocorticoid R/ H (IHC, IP, WB) NR3C2 TAO2 H (WB) M (WB) R (WB) X (WB) NGFI-B/Nur77/ H (IP, WB) Tec H (IHC, WB) M (IHC, WB) NR4A1

TOR H (FC, IHC, IP, WB) M (FC, IHC, IP, WB) R (IP, WB) H NGFI-B/NOR-1/ H (IP, WB) NR4A3 TTK H (IHC, WB) Nurr1/NGFI-B/ H (IHC, IP, WB) M (IHC, WB) Tyk2 H (WB) NR4A2 UBE2N/Ubc13 H PNR/NR2E3 H (IHC, WB) UBE2N (Ubc13)/ H Uev1a Complex PPAR/NR1C1 H (EMSA, IP, WB) PPAR/NR1C3 H (EMSA, IHC, IP, WB) M (WB) Ubiquitin H Pz Pl Y Ms H (IHC, WB) H Rb Ms PPAR/NR1C2 H (EMSA, WB) Ubiquitin/ H (IHC, WB) Ubiquitin+1 Progesterone R/ H (IHC, IP, WB) M (IHC, WB) R (IHC, WB) Pr (IHC, WB) H NR3C3 Ubiquitin+1 HH (ELISA, IHC, WB) H Progesterone R B/ H (IHC, IP, WB) Di-Ubiquitin/Ub2 H Ms NR3C3 Tri-Ubiquitin/Ub3 H PXR/NR1I2 H (IP, WB) Tetra-Ubiquitin/Ub4 H RAR/NR1B1 H (IP, WB) Penta-Ubiquitin/Ub5 H RAR/NR1B2 H (IP, WB) Hexa-Ubiquitin/Ub6 H RAR/NR1B3 H (IP, WB) Octa-Ubiquitin/Ub8 H Rev-erb A/NR1D1 H (IP, WB) Poly-Ubiquitin H Rev-erb/NR1D2 H (IHC, IP, WB) Uev1a (Mms2)/ H UBE2V1 ROR/NR1F1-3 H (IHC, IP, WB) ROR/NR1F1 H (IP, WB) VRK1 H (WB) ROR/RORC/NR1F3 H (FC, IP, WB) M (WB) WNK1 H (WB) M (WB) R (WB) H M R RORt/RORC2/NR1F3 H (FC) M (FC) Yes H (IHC, WB) M (IHC, WB) R (IHC, WB) H RXR/NR2B1 H (EMSA, IHC, IP, WB) ZAP70 HH (IHC, WB) H Z-Leu-Leu-Leu-CHO Ms RXR/NR2B2 H (IP, WB) (MG-132) RXR/NR2B3 H (IP, WB)

Nuclear Hormone Receptors SF-1/NR5A1 H (IHC, IP, WB)

Androgen R/NR3C4 H (FC, IHC, IP, WB) M (FC, IHC, WB) R (FC, IHC, WB) TLX/NR2E1 H (IP, WB)

CAR/NR1I3 H (IHC, IP, WB) M (WB) TR/NR1A1 H (IHC, WB)

COUP-TF I/NR2F1 H (IHC, IP, WB) TR1/NR1A2 H (IP, WB)

COUP-TF II/NR2F2 H (IHC, IP, WB) TR2/NR2C1 H (IP, WB)

78 For research use only. Not for use in diagnostic procedures. SIGNAL TRANSDUCTION

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

TR4/NR2C2 H (IHC, IP, WB) FKBP12.6 H (WB) M (WB) R (WB)

VDR/NR1I1 H (IHC, IP, WB) FKBP13 H (WB) M (WB) R (WB)

Phosphatases & Regulators FKBP25 H (WB) M (WB) R (WB)

Alkaline H (IP, WB) FKBP38 H (IHC, WB) M (IHC, WB) R (IHC, WB) Phosphatase/ALPI FKBP51 H (IHC, WB) M (IHC, WB) R (IHC, WB) Alkaline H M H (FC, IHC, WB) M (FC, IHC, IP, WB) R (FC, IHC) Phosphatase/ALPL FKBP52 H (IHC, WB) M (WB) R (WB)

Alkaline H (IHC, IP, WB) ILKAP/PP2C H (WB) M (WB) R (WB) Phosphatase/ALPP Laforin/EPM2A H (WB) Alkaline H (IP, WB) Phosphatase/ALPP/ LAR H (WB) M (WB) R (WB) ALPI LMW-PTP/ACP1 H (WB) M (WB) R (WB)

B220/CD45R M (CD, FC, IHC, IP) Lyp H (IHC, WB)

Calcineurin H MKP-3 H (WB) M (WB) R (WB)

Calcineurin A H (WB) M (WB) R (WB) OV-1 H (FC) R (FC) Calcineurin B H (WB) M (WB) R (WB) Phosphate Detection Ms Kit, Malachite Green CD45 H M H (FC, IHC) M (FA, FC, IHC, IP, WB) H PNUTS H (WB) M (WB) R (WB) CD45RO H (FC) PP1 H (WB) M (WB) R (WB) CDC14 H (WB) M (WB) R (WB) PP1 H (WB) CDC25A H (IHC, WB) M (IHC, WB) R (IHC, WB) PP1 Inhibitor-2 H (WB) M (WB) R (WB) CDC25B HH (IHC, WB) M (IHC, WB) R (IHC, WB) PP2C/PPM1A HH (WB) M (WB) R (WB) CDC25C H (WB) M (WB) R (WB) Cyclophilin A H PP2C/PPM1B H (WB) M (WB) R (WB) PP2C H (IHC, WB) Cyclophilin B H (WB) M (WB) R (WB) PP2C /PPM1L H (WB) M (WB) R (WB) Cyclophilin C H (WB) PP4 H (WB) DARPP-32 H (WB) M (IHC, WB) R (IHC, WB) PPP1R9B H (IHC, WB) R (IHC, WB) DEP-1/CD148 HH (FC, IP, WB) M (IP, WB) R (IP, WB) H PRG-1/LPPR4 H (WB) FKBP12 HH (WB) M (WB) R (WB) * UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization CD Cell Depletion ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection EMSA Gel Shift FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot s u mp

a an m ppoc u i

kDa H H

19 FKBP12.6

Phospho-RSK1/RSK2 in Mouse Cerebellum. Phosphorylated RSK1/RSK2 was Detection of Human FKBP12.6 by Western Blot. Western blot shows lysates of GRK2 in Alzheimer’s Disease Brain. G protein-coupled receptor kinase 2 detected in perfusion-fi xed frozen sections of mouse brain using a Rabbit Anti- human hippocampal tissue. The PVDF membrane was probed with a Goat Anti- (GRK2) was detected in immersion-fi xed paraffi n-embedded sections of human Human/Mouse/Rat Phospho-RSK1/RSK2 (RSK1 S221, RSK2 S227) Antigen Affi nity- Human/Mouse/Rat FKBP12.6 Biotinylated Antigen Affi nity-purifi ed Polyclonal Alzheimer’s disease brain using a Sheep Anti-Human/Mouse/Rat GRK2 Antigen purifi ed Polyclonal Antibody (Catalog # AF892). The tissue was stained with the Antibody (Catalog # BAF4174) followed by a HRP-conjugated Anti-Goat IgG Affi nity-purifi ed Polyclonal Antibody (Catalog # AF4339). Before incubation with Anti-Rabbit HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS005; brown) and Secondary Antibody (Catalog # HAF109). FKBP12.6 was detected at approximately the primary antibody, the tissue was subjected to heat-induced epitope retrieval counterstained with hematoxylin (blue). Specifi c staining was localized to Purkinje 13 kDa (as indicated). using Antigen Retrieval Reagent-Basic (Catalog # CTS013). The tissue was stained cells in the cerebellum. using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (Catalog # CTS019; brown) and counterstained with hematoxylin (blue).

www.RnDSystems.com/go/SignalTransduction 79 SIGNAL TRANSDUCTION

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Phosphatases & Regulators, continued EphA10 HH (IHC)

PRL/PTP4A H (IHC) EphB1 RR (FC, IHC, WB)

PRL-3 H (WB) M (WB) R (WB) EphB2 H M H (FC, IHC, WB) M (FC, IHC, WB)

Prostatic Acid HH (IHC, WB) EphB3 H M H (WB) M (FC, IHC, WB) Phosphatase/ACPP EphB4 H M H (FC, IHC, WB) M (FC, IHC, WB) H PTP1B HH (IHC, WB) M (WB) R (WB) H M R EphB6 H M H (FC, WB) M (WB) Oxidized PTP Active H (WB) M (WB) R (WB) Ms (WB) Site ErbB2/Her2 HH (B/N, ELISA, FC, IHC, WB) M (FC, IHC, WB) H

PTP-MEG2 H (IHC, WB) M (IHC, WB) R (IHC, WB) ErbB3/Her3 H M H (B/N, ELISA, FC, IHC, WB) M (IHC, WB) H

PTPN13/PTPL1 H (WB) ErbB4/Her4 H M H (FC, WB) H

PTPN14 H (IHC, WB) FGF R1-4 H (IHC, WB)

PTPR H (IHC, WB) M (WB) R (WB) FGF R1 HH (B/N, WB) H

PTPR H (IHC, WB) FGF R1 H

PTPRM H (WB) M (WB) R (WB) FGF R1 H

PTPRT H (WB) FGF R2 H M H (B/N, FC, IHC, WB) M (B/N, WB) H

PTPRZ H (IHC, WB) FGF R2 H M H (WB) H Inorganic H FGF R2 H M Pyrophosphatase/ PPA1 FGF R3 H M H (B/N, FC, IHC, WB) M (B/N) H Serine/Threonine Ms FGF R4 HH (FC, IHC, WB) M (IHC, WB) H Phosphatase FGF R5/FGFRL1 MH (WB) M (IHC, WB) Substrates Flt-3/Flk-2 H M H (FC, IHC, WB) M (FC, IHC, WB) H SHIP H (IHC, WB) M (IHC, WB) R (IHC, WB) HGF R/c-MET H M Ca H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M Ca SHP-1 HH (IHC, WB) M (IHC, WB) R (IHC, WB) H M R Ca (B/N, ELISA, WB)

SHP-2 HH (IHC, WB) M (IHC, WB) R (WB) H M R IGF-I R H M H (B/N, ELISA, FC, IHC, WB) M (B/N, FC, IHC, WB) H

TC-PTP HH (WB) M (WB) R (WB) H M R IGF-II R HH (B/N, ELISA, FC, IHC, WB) H

TRACP/PAP/ACP5 HH (IP, WB) Insulin R/CD220 HH (FC, IHC, WB) M (FC) H

Tyrosine Ms INSRR H (FC, IHC, WB) Phosphatase Substrate I M-CSF R H M H (B/N, ELISA, FC, IHC, WB) M (FC, IHC, WB) H

VHR HH (IHC, WB) M (WB) R (WB) Mer H M H (ELISA, FC, WB) M (WB) H

Receptor Tyrosine Kinases MSP R/Ron H M H (B/N, FC, WB) M (B/N, WB) H

ALK/CD246 HH (WB) MuSK H (WB) R (B/N, WB)

Axl H M H (B/N, ELISA, FC, IHC, WB) M (ELISA, FC, WB) H M PDGF R H M H (B/N, FC, IHC, IP, WB) M (B/N, IHC, WB) H M

c-Abl H (WB) PDGF R H M H (B/N, FC, IHC, IP, WB) M (IHC, WB) H M

CCK4 H (IHC, WB) M (WB) R (IHC, WB) Ret H M H (FC, IHC, WB) M (IHC, WB) H

DDR1 H M H (IHC, IP, WB) H ROR1 Receptor H (FC, WB) H Tyrosine Kinase DDR2 HH (FC, IHC, WB) H RTK-like Orphan H (FC, WB) H Dtk H M H (ELISA, FC, IHC, WB) M (ELISA, FC, IHC, WB) H M Receptor 2/ROR2 EGF R/ErbB1 H M H (ELISA, FC, IHC, IP, WB) M (IHC, WB) H RTK Array Kit

Eph H M R Ryk H (WB) M (IHC, WB)

EphA1 H M H (FC, IHC, WB) M (FC, IHC, WB) H SCF R/c-kit HH (B/N, ELISA, FC, IHC, WB) M (FC, IHC, WB) H

EphA2 H M H (FC, IHC, WB) M (FC, IHC, WB) H STYK1 H (FC, IHC, WB)

EphA3 H M M (IHC, WB) Tie-1 HH (FC, IHC, WB) H

EphA4 MM (IHC, WB) Tie-2 H M R Z H (B/N, ELISA, FC, IHC, WB) M (B/N, FC, IHC, WB) H M Z (B/N, IHC, WB) EphA5 H M R H (WB) M (FC, WB) R (FC, IHC, WB) H M TrkA H R H (B/N, FC, IHC, WB) M (IHC, WB) R (IHC, WB) H R EphA6 H M M (B/N, WB) TrkB H M H (FC, IHC, WB) M (B/N, IHC, WB) H EphA7 H M M (WB) TrkC H M H (B/N, FC, IHC, WB) M (B/N, IHC, WB) H EphA8 MM (WB) VEGF R1, R2, R3 H (FC)

80 For research use only. Not for use in diagnostic procedures. SIGNAL TRANSDUCTION

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

VEGF R1/Flt-1 H M H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M DACH2 H (WB) M (WB)

VEGF R2/KDR/Flk-1 H M H (B/N, ELISA, FC, IHC, WB) M (B/N, ELISA, FC, IHC, WB) H M DC-SCRIPT/ZNF366 H (FC, WB)

VEGF R3/Flt-4 H M H (ELISA, FC, IHC, WB) M (ELISA, FC, WB) H M DEC2 H (WB)

Transcription Factors & Regulators DDX5 H (WB) M (WB)

ACLP H (FC) DEK H (IHC, WB)

ADNP H (IHC, WB) M (IHC, WB) R (IHC, WB) DIDO1 H (WB)

AHR H (IHC, WB) M (IHC, WB) DLX5 H (IHC)

AP-2 H (WB) DNMT1 H (IHC, WB)

AP-2 H (IHC, WB) DNMT3A H (IHC, WB) M (WB)

AP-2 H (WB) E2F-1 H (IHC, WB)

ARA54 H (WB) E2F-2 H (WB)

ARNT/HIF-1 H (IHC, WB) E2F-4 H (WB)

ARX H (WB) EBF-1 H (WB) M (WB)

ASCL1/Mash1 M (IHC, WB) EBF-2 H (IHC, WB) M (IHC, WB)

ASCL2/Mash2 H (IHC, WB) EBF-3 H (WB) M (WB)

ATF1 H (WB) M (WB) EED H (WB)

ATF2 H (IHC, WB) M (WB) R (IHC, WB) EGR1 H (IHC, WB)

ATN1 H (IHC, WB) M (IHC, WB) Elk-1 H (WB) M (WB) R (WB) Ca (WB) Pr (WB) Z (WB)

Autoimmune H (FC, WB) M (FC, WB) EMX2 H (WB) M (WB) Regulator/AIRE Engrailed-2 H (IHC, WB) BACH1 H (IHC, WB) M (WB) EOMES H (FC, IHC, WB) BASP1 H (WB) ETV5 H (IHC) Bcl-6 H (FC, IHC, WB) M (FC, IHC, WB) EZH2 H (IHC, WB) M (IHC, WB) Bcl-9 H (WB) FHL1 H (WB) BLIMP1/PRDM1 H (FC, WB) M (FC, WB) FHL2 H (WB) Brachyury H (ChIP, FC, IHC, WB) M (ChIP, FC, IHC, WB) H FLI1 H (WB) Brg1 H (WB) FosB/G0S3 H (IHC, WB) M (WB) BTF3 H (IHC, WB) M (IHC, WB) R (IHC, WB) FoxC1 H (WB) C1D H (WB) FoxC2 H (IHC, WB) M (IHC) CBP H (IHC, WB) M (IHC, WB) R (IHC, WB) FoxF1 H (WB) M (WB) CDX2 H (IHC, WB) FoxF2 H (WB) CDX4 H (IHC, WB) FoxH1 H (IHC, WB) CEBP  H (IHC, WB) FoxJ3 H (WB) M (WB) CHD1 H (IHC, WB) M (WB) FoxK1 H (IHC, WB) CIS-1 H (IHC, WB) FoxM1 H (IHC, WB) Cited-2 H (WB) M (WB) FoxO1/FKHR H (IHC, WB) CREB H (ChIP, IHC, WB) M (WB) R (WB) H M R FoxO3 H (WB) M (WB) CREG H M H (WB) M (WB) FoxP1 H (WB) CRX H (WB) FoxP2 H (IHC, WB) M (IHC, WB) CSL H (IHC, WB) FoxP4 H (WB) CtBP1 H (WB) FRA-1 H (WB) CTCF H (WB) GATA-1 H (FC, IHC, WB) M (WB) Cyclin A2 H (WB) M (WB) GATA-2 H (FC, IHC, WB) M (WB) Cyclin B1 H (WB) M (WB) GATA-3 H (FC, IHC, WB) M (IHC, WB) Cyclin B2 H (IHC, WB) M (WB) GATA-4 H (ChIP, IHC, WB) H Cyclin D3 H (IHC, WB) GATA-5 H (ChIP, IHC, WB) H * UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot

www.RnDSystems.com/go/SignalTransduction 81 SIGNAL TRANSDUCTION

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Transcription Factors & Regulators, continued Islet-2 H (IHC, WB)

GATA-6 H (ChIP, IHC, WB) H Jade-1/PHF17 H (IHC, WB)

GBX2 H (WB) Jumonji/JARID2 H (IHC, WB) M (WB)

GFI-1 H (IHC, WB) JunB H (WB)

GLI-1 H (ChIP, IHC, WB) M (IHC, WB) H c-Jun H (IHC, WB) M (IHC, WB)

GLI-2 H (ChIP, IHC, WB) M (IHC, WB) H JunD H (WB) M (WB)

GLI-3 H (ChIP, IHC, WB) M (ChIP, IHC, WB) H Keap1 H (WB) M (WB) R (WB)

Goosecoid H (IHC, WB) KLF2 H (IHC, WB) M (IHC, WB) R (IHC, WB)

Hairless H (IHC, WB) KLF4 H (ChIP, FC, IHC, WB) M (ChIP, IHC, WB) H M

HAND1 H (IHC, WB) KLF5 H (IHC, WB)

HAND2 H (IHC, WB) M (IHC, WB) KLF6 H (WB)

Host Cell Factor 1/ H (WB) KLF10 H (WB) HCFC1 KLF17 H (WB) HES-1 H (IHC, WB) LEDGF HH (IHC, WB) HES-4 H (IHC, WB) LIM1 H (IHC, WB) HIF-1 H (ChIP, FC, IHC, IP, WB) M (ChIP, FC, IHC, IP, WB) H M R (ChIP, IP, WB) LITAF H (WB)

HIF-2/EPAS1 H (ChIP, IHC, WB) M (IHC, WB) R (ChIP, IHC, WB) H LMO2 H (IHC, WB)

Histone Deacetylase HH (ChIP, WB) M (ChIP, WB) LMO4 H (WB) M (WB) 8/HDAC8 LPP H (WB) M (WB) Histone H3 H Lysine (K)-specifi c H (WB) Histone H4 H (IHC, WB) M (IHC, WB) H Demethylase 3A/ KDM3A HMGA1B H (WB) M (WB) Lysine (K)-specifi c H (IHC, WB) HMGA2 H (IHC, WB) M (IHC, WB) Demethylase 5B/ KDM5B HMGB1/HMG-1 HH (ChIP, FC, IHC, WB) H MafB H (WB) R (WB) HMGB3 H (IHC, WB) M (IHC, WB) MafF H (IHC, WB) HNF-3/FoxA1 H (IHC, WB) MafG H (WB) HNF-3/FoxA2 H (ChIP, IHC, WB) MafK H (WB) M (WB) HNF-6/ONECUT1 H (IHC, WB) Max H (WB) HOXA1 H (IHC, WB) MBD4 H (WB) M (WB) HOXB1 H (IHC) MCM2 H (IHC, WB) M (IHC, WB) HSF2 H (WB) M (WB) MED4 H (WB) HSF4 H (WB) M (WB) MEF2C H (WB) ICAT H (WB) MITF H (IHC, WB) ID1 H (WB) M (WB) Miz-1/ZBTB17 H (WB) ID2 H (IHC, WB) MLX H (IHC, WB) Ikaros H (ChIP, FC, IHC, WB) H MSX1 H (WB) M (WB) ING1 H (IHC, WB) M (WB) MTF2 H (IHC) IRF1 H (WB) M (WB) Mxi1 H (IHC) IRF2 H (WB) M (WB) MYB H (WB) M (WB) IRF3 H (FC, IHC, WB) M (WB) c-Myc H (ChIP, IHC, WB) H IRF4 H (WB) M (WB) MYCL1/L-Myc H (IHC, WB) M (IHC, WB) IRF5 H (FC, IHC, WB) Myocardin H (WB) M (WB) IRF6 H (WB) M (WB) MyoD H (IHC) IRF8 H (IHC, WB) Myogenin H (IHC) M (IHC) IRF9 H (WB) M (WB) Nanog H (ChIP, FC, IHC, WB) M (IHC, WB) IRF2BP1 H (IHC, WB) NCOR1 H (WB) M (WB) R (WB) Islet-1 H (IHC, WB)

82 For research use only. Not for use in diagnostic procedures. SIGNAL TRANSDUCTION

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

NCOR2 H (IHC) p53 HH (ChIP, FC, IHC, IP, WB) M (ChIP, IP, WB) R (ChIP, IP, WB) H M

NeuroD1 H (IHC, WB) M (IHC, WB) p63/TP73L H (IHC, WB)

NeuroD2 H (WB) M (WB) p15INK4b/CDKN2B H (WB)

Neurogenin-1 H (WB) p16INK4a/CDKN2A H (WB)

Neurogenin-2 H (IHC) R (IHC) p300 H (ChIP, IHC, WB) H

Neurogenin-3 H (WB) PA2G4 H (WB)

NFATC1 H (WB) Park7/DJ-1 H (IHC, WB) M (WB) H

NFATC2 H (WB) Pax2 H (IHC, WB)

NFATC3 H (WB) M (WB) R (WB) Pax3 H (FC, IHC, WB) M (FC, IHC, WB)

NKX2.5 H (IHC, WB) Pax4 H (IHC, WB)

NKX3.1 H (IHC, WB) M (WB) Pax5/BSAP H (IHC, WB)

NKX6.1 H (IHC, WB) M (IHC, WB) Pax6 M (IHC) R (IHC) Ch (IHC)

Nrf1 H (WB) M (WB) R (WB) Pax7 H (IHC, WB) M (IHC) R (IHC) Ch (IHC)

Nrf2 H (WB) M (WB) R (WB) PDX-1/IPF1 H (FC, IHC, WB) M (FC, IHC, WB) R (IHC, WB)

NRL H (WB) PGC1 H (WB)

OASIS/CREB3L1 H (WB) PGC1 H (WB) M (WB)

Oct1 H (IP, WB) M (IP, WB) PHOX2B H (IHC) M (IHC)

Oct-3/4 H (ChIP, FC, IHC, IP, WB) M (FC, IHC, WB) H M PIAS2 H (WB)

Oct-4A H (FC, IHC, WB) PIWIL4 H (IHC)

Olig 1, 2, 3 H (FC, IHC) PIWIL1/HIWI H (IHC) M (IHC)

Olig1 H (IHC, WB) M (IHC, WB) PIWIL2 H (WB) R (WB)

Olig2 H (ChIP, IHC, WB) H PLZF H (FC, IHC, WB)

Olig3 H (IHC, WB) M (IHC, WB) PRDM14 H (IHC, WB)

ONECUT2/OC-2 H (IHC, WB) PRDM16 H (IHC, WB) M (IHC, WB)

Otx2 H (IHC, WB) PREB H (WB) * UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot x te or C

e s u

kDa Mo

NeuroD2 37

Pax6 in Undiﬀ erentiated Rat Cortical Stem Cells. Pax6 was detected in Detection of Mouse NeuroD2 by Western Blot. Western blot shows lysates of Olig1 in Human Glioma. Olig1 was detected in immersion-fi xed paraffi n- immersion-fi xed undiff erentiated rat cortical stem cells using a Mouse Anti-Mouse/ mouse cortical tissue. The PVDF Membrane was probed with a Goat Anti-Human/ embedded sections of human glioma using a Mouse Anti-Human Olig1 Monoclonal Rat/Chicken Pax6 Monoclonal Antibody (Catalog # MAB1260). The cells were stained Mouse NeuroD2 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF2775) Antibody (Catalog # MAB2417). Tissue was stained with the Anti-Mouse HRP-DAB using the NorthernLights™ 557-conjugated Donkey Anti-Mouse IgG Secondary followed by a HRP-conjugated Chicken Anti-Goat IgG Secondary Antibody (Catalog # Cell & Tissue Staining Kit (Catalog # CTS002; brown) and counterstained with Antibody (Catalog # NL007; yellow) and counter stained with DAPI (blue). HAF019). NeuroD2 was detected at approximately 41 kDa (as indicated). hematoxylin (blue). Specifi c staining was localized to the nuclei of glial cells.

www.RnDSystems.com/go/SignalTransduction 83 SIGNAL TRANSDUCTION

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Transcription Factors & Regulators, continued Sp3 H (WB)

Prohibitin 2 H (WB) M (WB) R (WB) STAT1 H (FC, IHC, IP, WB) M (FC, IP, WB) H M

Prox1 H (IHC, WB) STAT2 H (FC, IHC, IP, WB) H

PTF1A H (IHC, WB) STAT3 H (ChIP, FC, IHC, IP, WB) M (ChIP, FC, IHC, IP, WB) H M R (ChIP, FC, IHC, IP, WB) PU.1/Spi-1 H (FC, IHC, WB) M (WB) STAT4 H (ELISA, IP, WB) M (ELISA, IP, WB) H Pygopus-1 H (WB) M (WB) STAT5a/b H (ChIP, FC, IHC, WB) M (ChIP, WB) H M Pygopus-2 H (IHC, WB) STAT5a H (FC, IHC, IP, WB) M (IHC, IP, WB) RAP80 H STAT5b H (FC, IP, WB) M (FC, IP, WB) RCOR1/CoREST H (IHC, WB) M (WB) STAT6 H (FC, IHC, IP, WB) M (FC, IP, WB) R (FC, WB) H M Rex-1/ZFP42 H (IHC, WB) SUZ12 H (IHC, WB) RFC1 H (WB) TACC3 H (WB) RUNX1/CBFA2 H (WB) M (WB) R (WB) TAF5L H (WB) RUNX2/CBFA1 H (ChIP, IHC, WB) M (IHC) H T-bet/TBX21 H (FC, IHC, WB) H RUNX3/CBFA3 H (FC, IHC, WB) M (FC, IHC, WB) TBX2 H (IHC, WB) SALL1 H (IHC, IP, WB) TBX3 H (IHC, WB) SALL4 H (IHC, WB) M (IHC, WB) TBX5 H (WB) M (WB) SCL/Tal1 H (IHC, WB) TBX6 H (IHC, WB) SIN3A H (WB) M (WB) TBX18 H (IHC, WB) Sirtuin 2/SIRT2 HH (IP, WB) TCF-2/HNF-1 H (IHC, WB) Sirtuin 5/SIRT5 H (IP, WB) TCF-3/E2A H (IHC, WB) SKI H (IHC, WB) TCF-12/HTF4 H (IHC, WB) Smad1 H (IHC, WB) TCF7/TCF1 H (IHC, WB) Smad2 H (ChIP, IHC, WB) M (ChIP, WB) H M R TCF7L1/TCF3 H (IHC, WB) M (WB) Smad2/3 H (ChIP, WB) M (ChIP, WB) H M R Teneurin-1 H (WB) Smad3 H (ChIP, FC, IHC, WB) M (ChIP, WB) H M R Teneurin-2 H (WB) M (WB) R (WB) Smad4 H (ChIP, FC, IHC, WB) H Teneurin-4 H (WB) M (WB) R (WB) Smad5 H (IHC, WB) M (WB) TFCP2L1 H (WB) Smad7 H (IHC, WB) M (IHC, WB) R (IHC, WB) THAP11 H (IHC, WB) M (IHC, WB) Smad8 H (IHC, WB) Th-POK H (WB) SMARCA5/SNF2H H (IHC) TLE1 H (WB) SMURF2 H (WB) M (WB) R (WB) TLE3 H (WB) M (WB) Snail H (ChIP, IHC, WB) H TOP2A H (WB) SOCS-1 H (IP, WB) M (IP, WB) TOP2B H (IHC, WB) M (IHC, WB) SOCS-2 H (WB) TORC1 M (IHC) SOCS-3 H (IHC, WB) M (IHC, WB) TORC2 H (WB) M (WB) SOCS-4 H (WB) M (WB) TORC3 M (WB) SOX1 H (IHC, WB) M (WB) TRIM21 H (IHC, WB) SOX2 H (ChIP, FC, IHC, WB) M (FC, IHC, WB) H M TRIM32 H (WB) M (WB) SOX3 H (FC, IHC, WB) TRPS1 H (WB) SOX5 H (WB) TSC22 M (WB) R (WB) SOX7 H (FC, IHC, WB) Twist-1 H (FC, IHC) SOX9 H (IHC, WB) Twist-2 H (IHC) SOX10 H (IHC, WB) R (IHC) UTF1 H (IHC, WB) SOX15 H (IHC, WB) WDR5 H (IHC, WB) M (IHC, WB) SOX17 H (ChIP, FC, IHC, WB) M WT1 H (FC, WB) SOX18 H (WB) XBP1 H (FC, WB) SOX21 H (IHC, WB) YY1 H (WB)

84 For research use only. Not for use in diagnostic procedures. SIGNAL TRANSDUCTION

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

ZBTB38 H (WB) Caveolin-3 H (IHC, WB)

ZBTB7A/Pokemon H (WB) M (WB) cGMP Ms

ZEB1 H (FC, IHC) Cortactin H (WB) R (WB)

ZIC1 H (IHC, WB) M (IHC, WB) CRABP2 H (WB)

ZIC3 H (IHC, WB) M (IHC, WB) CRF21 H (WB)

ZNF24 H (IHC, WB) CSRP1 H (WB) M (WB) R (WB)

ZNF143 H (IHC) CSRP2 H (WB)

ZNF206 H (IHC, WB) Cytohesin-1 H (WB)

ZNF281 H (WB) M (WB) DIAPH-2 H (WB)

Translational Regulators Eps8 H (WB)

eEF-2 H (WB) M (WB) R (WB) GMF- H (IHC, WB)

eIF2 H (WB) HCLS1 H (WB) M (WB) R (WB)

eIF4B H (WB) HR23A/Rad23A H

eIF4E H (IHC, WB) M (IHC, WB) R (IHC, WB) HSP27 HH (WB) M (WB) R (WB) H M R

eIF4G1 H (WB) IMP2 H (WB) M (WB) R (WB)

GCN2 H (WB) IQGAP1 H (WB)

MBD3 H (WB) IQGAP2 H (WB)

PDCD4 H (IHC) ISG15/UCRP HH (IHC, WB) H Ms

PERK H (WB) JAB1 H (WB) M (WB) R (WB)

Ribosomal Protein S6 H (IHC, WB) M (IHC, WB) R (IHC, WB) H M R JIP1 H (WB) M (WB) R (WB)

Additional Signal Transduction Molecules LARG H (WB)

4-Hydroxynonenal Ms (WB) LAX1 H (FC, WB)

Actin H (IHC, WB) M (IHC, WB) R (IHC, WB) MARCH8 H (IHC, WB) M (IHC, WB) R (IHC, WB)

AFAP H (WB) MBP H (WB) M (WB) R (WB) B (WB)

Ajuba H (WB) MP1 H (WB) M (WB) R (WB)

AMSH/STAMBP H (WB) Myosin Heavy Chain H (FC, IHC)

APC H (IHC, WB) NEDD4 H (WB) M (WB) R (WB)

APLP-1 H (FC, IHC, IP, WB) M (IP, WB) NEDD8 HH (IHC, WB) M (IHC, WB) R (IHC, WB) H Ms

APLP-2 H (WB) M (WB) Nitrotyrosine Ms (IHC, WB)

APP/ HH (IHC, IP, WB) M (IHC, WB) R (WB) Ca (WB) Ch (WB) H M R NRAGE H (FC, IHC, WB) M (WB) R (WB) Protease Nexin II Pr (WB) Ms (WB) Palladin H (IHC, WB) APP 695+1 H (IHC, IP, WB) PDZD11 H (WB) APP+1 HH (WB) PDZK1 H (IHC, WB) M (IHC, WB) R (WB) ARHGAP1 H (WB) M (WB) R (WB) PLA2G2A H M H (IP, WB) M (IHC, WB) -Arrestin 1 H (IHC, WB) PLA2G4A H -Arrestin 2 H (IHC) PLC-1 H (WB) M (WB) BSRP-A H (WB) M (IHC, WB) PLC-3 H (WB) BSRP-C M (IHC, WB) PLC-4 H (WB) M (WB) R (WB) cAMP Ms (IHC) Ms PLC-1 H (WB) M (WB) R (WB) Carbonic Anhydrase H VII/CA7 PLC-2 H (IHC, WB) M (IHC, WB)

Carboxymethyl Ms (WB) PLC-3 H (WB) M (WB) Lysine PLD1 H (WB) Caveolin-1 H (IHC, WB) M (WB) R (WB) PRR5 H (WB) Caveolin-2 H (IHC, WB) M (IHC, WB) R (IHC, WB) RAB25 H (WB) * UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot

www.RnDSystems.com/go/SignalTransduction 85 SIGNAL TRANSDUCTION

RECOMBINANT RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs/UPP* MOLECULE PROTEINS ANTIBODIES ELISAs/UPP*

Additional Signal Transduction Molecules, continued SEZ6L H (WB) M (IHC, WB)

RACK1 H (IHC, WB) M (IHC, WB) R (IHC, WB) SKAP55 H (WB)

Rad23 H (WB) M (WB) R (WB) Y (WB) SLAP-130 H (FC, WB)

RalA/RalB H (WB) M (WB) R (WB) SOS2 H (WB)

RalA H (WB) M (WB) R (WB) SPRED1 H (WB)

RalB H (WB) SPRED2 H (IHC, WB)

Rap1A/B H (IHC, WB) M (IHC, WB) R (IHC, WB) SPRY1 H (WB)

Rap2A/B H (WB) M (WB) R (WB) SPRY2 H (WB) M (WB)

Rap2B H (WB) SPRY3 H (WB)

Ras H (WB) M (WB) R (WB) SPRY4 H (WB)

M-Ras/R-Ras3 H (WB) R (WB) STIM1 H (WB) M (WB) R (WB)

RASSF2 H (WB) M (WB) TAB1 H (IHC, WB) M (IHC, WB)

RGS8 H (IHC, WB) TC21/R-Ras2 H (IHC, WB) R (IHC, WB)

Rheb H (IHC, WB) M (IHC, WB) R (IHC, WB) TIAM1 H (WB) M (WB)

Rictor H (WB) TOB1 H (WB)

Rit1 H (WB) TSC1 H (FC, WB) M (FC, WB) R (FC, WB)

Rit2 H (IHC, WB) TSC2 H (IHC, WB) M (IHC, WB)

RKIP H (WB) M (WB) R (WB) Tyrosine Ms (FC, IHC, IP, WB)

RND3 H (WB) M (WB) UBE2K/E2-25K HH (IHC, WB) M (IHC, WB) R (WB)

RPTOR H (WB) M (WB) Vinculin H (IHC, WB) R (WB)

RTN1-A/NSP R (IHC) WASF1/WAVE1 H (WB) M (WB)

SCAMP3 H (WB) M (WB) WASF3/WAVE3 H (WB) M (WB)

SDPR H (WB) M (WB) Zyxin H (IHC, WB)

Secretagogin H (WB) M (WB) R (WB) * UPP UBIQUITIN PROTEASOME PATHWAY ASSAY KITS & REAGENTS Species Key: H Human M Mouse R Rat B Bovine Ba Bacterial Ca Canine Ch Chicken CR Cotton Rat D Drosophila E Equine F Feline GP Guinea Pig Ms Multispecies Pl Plant P Porcine Pr Primate Pz Protozoa Rb Rabbit RM Rhesus/Macaque V Viral X Xenopus Y Yeast Z Zebrafi sh Application Key: B/N Blocking/Neutralization ChIP Chromatin Immunoprecipitation ELISA ELISA Capture and/or Detection FA Functional Assay FC Flow Cytometry IF Immunofl uorescence IHC Immunohistochemistry IP Immunoprecipitation WB Western blot

A. B. ain Y Br 5 e s u SH-SY kDa Mo kDa 93 116 65 BSRP-A 95 40 SPRY2

53 24 37 18

6 19

SHANK2 in Human Cerebellum. SH3 and multiple ANKyrin repeat domain protein Detection of Mouse BSRP-A and Human SPRY2 by Western Blot. Western blots SEZ6L in Mouse Hippocampus. Seizure 6-Like protein (SEZ6L) also known as 2 (SHANK2) was detected in immersion-fi xed paraffi n-embedded sections of show lysates of mouse brain tissue and the SH-SY5Y human neuroblastoma cell line. Brain-specifi c receptor-like protein B (BSRP-B) was detected in perfusion-fi xed human brain using a Mouse Anti-Human SHANK2 Monoclonal Antibody (Catalog # A. The PVDF membrane was probed with a Rat Anti-Mouse BSRP-A Monoclonal frozen sections of mouse brain using a Sheep Anti-Mouse SEZ6L Antigen Affi nity- MAB7035). Before incubation with the primary antibody, the tissue was subjected Antibody (Catalog # MAB4916) followed by a HRP-conjugated Goat Anti-Rat IgG purifi ed Polyclonal Antibody (Catalog # AF4804). The tissue was stained using the to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog Secondary Antibody (Catalog # HAF005). B. The PVDF Membrane was probed with NorthernLights™ 557-conjugated Donkey Anti-Sheep IgG Secondary Antibody # CTS013). The tissue was stained using the Anti-Mouse HRP-DAB Cell & Tissue a Goat Anti-Human/Mouse SPRY2 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # NL010; red) and counterstained with DAPI (blue). Specifi c staining was Staining Kit (Catalog # CTS002; brown) and counterstained with hematoxylin (blue). (Catalog # AF6157) followed by a HRP-conjugated Donkey Anti-Goat IgG Secondary localized to neuronal cell bodies and processes in the hippocampus. Specifi c staining was localized to Purkinje neurons in the cerebellum. Antibody (Catalog # HAF109). BSRP-A and SPRY2 were detected at approximately 100 kDa and 35 kDa, respectively (as indicated).

86 For research use only. Not for use in diagnostic procedures. SPECIALIZED TOOLS

ANTIBODY ARRAYS & MULTIPLEX ASSAYS Antibody Arrays: Proteome Proﬁ ler™ R&D Systems Proteome Profi ler Arrays are screening tools that simultaneously detect the relative levels of multiple proteins and/or phosphorylated proteins in each sample. Each kit contains buff ers, detection conjugates, and nitrocellulose membranes spotted with a carefully selected panel of specifi c capture antibodies. Human Angiogenesis Array PRODUCT DESCRIPTION CATALOG # SIZE Human Angiogenesis Array Kit Contains 4 arrays - each spotted in duplicate with 55 diff erent angiogenesis antibodies ARY007 1 Kit

Activin A, ADAMTS1, Angiogenin, Angiopoietin-1, Angiopoietin-2, Angiostatin/Plasminogen, Amphiregulin, Artemin, Coagulation Factor III/TF, CXCL8/IL-8, CXCL16, DPPIV/CD26, EGF, EG- VEGF, Endoglin, Endostatin/Collagen XVIII, Endothelin-1, FGF acidic, FGF basic, FGF-4, FGF-7/KGF, GDNF, GM-CSF, HB-EGF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IL-1/IL-1F2, LAP (TGF-1), Leptin, MCP-1/CCL2, MIP-1/CCL3, MMP-8, MMP-9, NRG1-1/HRG1-1, Pentraxin 3, PD-ECGF, PDGF-AA, PDGF-AB/BB, Persephin, PF4/CXCL4, PlGF, Prolactin, Serpin B5, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, TIMP- 4, Thrombospondin-1, Thrombospondin-2, uPA, Vasohibin, VEGF, VEGF-C

Mouse Angiogenesis Array PRODUCT DESCRIPTION CATALOG # SIZE Mouse Angiogenesis Array Kit Contains 4 arrays - each spotted in duplicate with 53 diff erent angiogenesis antibodies ARY015 1 Kit

ADAMTS1, Amphiregulin, Angiogenin, Angiopoietin-1, Angiopoietin-3, Coagulation Factor III/TF, CXCL16, Cyr61/CNN1, DLL4, DPPIV/CD26, EGF, Endoglin/CD105, Endostatin/Collagen XVIII, Endothelin-1, FGF acidic, FGF basic, FGF-7/KGF, Fractalkine, GM-CSF, HB-EGF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IL-1, IL-1/IL-1F2, IL-10, IP-10/CRG-2/CXCL10, JE/MCP-1/CCL2, KC/CXCL1, Leptin, MIP-1/CCL3, MMP-3 (pro and mature), MMP-8 (pro), MMP-9 (pro and active), NOV/CCN3/IGFBP-9, Osteopontin, PD-ECGF, PDGF-AA, PDGF-AB, PDGF-BB, Pentraxin-3, PF4/CXCL4, PlGF-2, Prolactin, Proliferin, SDF-1/CXCL12, Serpin E1/PAI-1, Serpin F1/PEDF, Thrombospondin-2,TIMP-1, TIMP-4, VEGF, VEGF-B

Human Apoptosis Array PRODUCT DESCRIPTION CATALOG # SIZE Human Apoptosis Array Kit Contains 4 arrays - each spotted in duplicate with 35 diff erent apoptosis-related antibodies ARY009 1 Kit

Bad, Bax, Bcl-2, Bcl-x, Pro-Caspase-3, Cleaved Caspase-3, Catalase, Claspin, Clusterin, Cytochrome c, FADD, Fas/TNFSF6, HIF-1, HO-1/HMOX1/HSP32, HO-2/HMOX2, HSP27, HSP60, HSP70, HTRA2/Omi, cIAP-1, cIAP-2, Livin, p21/CIP1/CDNK1A, p27/Kip1, PON2, Phospho-p53 (S15), Phospho-p53 (S46), Phospho-p53 (S392), Phospho-Rad17 (S635), SMAC/Diablo, Survivin, TNF RI/TNFRSF1A, TRAIL R1/DR4, TRAIL R2/DR5, XIAP

Human Cell Stress Array PRODUCT DESCRIPTION CATALOG # SIZE Human Cell Stress Array Kit Contains 4 arrays – each spotted in duplicate with 26 diff erent cell stress-related antibodies ARY018 1 Kit

ADAMTS1, Bcl-2, Carbonic Anhydrase IX, Cited-2, COX-2, Cytochrome c, Dkk-4, FABP1/L-FABP, HIF-1, HIF-2, Phospho-HSP27 (S78/S82), HSP60, HSP70, IDO, Phospho-JNK Pan (T183/Y185), NFB1, p21/CIP1, p27/Kip1, Phospho-p38 (T180/Y182), Phospho-p53 (S46), PON1, PON2, PON3, Thioredoxin-1, SIRT2, SOD2

Human Cytokine Array, Panel A PRODUCT DESCRIPTION CATALOG # SIZE Human Cytokine Array Kit, Panel A Contains 4 arrays - each spotted in duplicate with 36 diff erent cytokine antibodies ARY005 1 Kit

C5a, CD40 Ligand/CD154, CXCL8/IL-8, G-CSF, GM-CSF, GRO/CXCL1, I-309/CCL1, ICAM-1/CD54, IFN-, IL-1, IL-1/IL-1F2, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p70, IL-13, IL-16, IL-17, IL-17E, IL-23, IL-27, IL-32,IP-10/CRG-2/CXCL10, I-TAC/CXCL11, MCP-1/CCL2, MIF, MIP-1/CCL3,MIP-1/CCL4,RANTES/CCL5, SDF-1/CXCL12, Serpin E1/PAI-1, TNF-,TREM-1

Mouse Cytokine Array, Panel A PRODUCT DESCRIPTION CATALOG # SIZE Mouse Cytokine Array Kit, Panel A Contains 4 arrays - each spotted in duplicate with 40 diff erent cytokine antibodies ARY006 1 Kit

BLC, C5a, Eotaxin/CCL11, G-CSF, GM-CSF, I-309/CCL1, ICAM-1/CD54, IFN-, IL-1, IL-1/IL-1F2, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 p70, IL-13, IL-16, IL-17, IL-23, IL-27, IP-10/CRG-2/CXCL10, I-TAC/CXCL11, KC, M-CSF, JE/CCL2/MCP-1, MCP-5/CCL12, MIG/CXCL9, MIP-1/CCL3,MIP-1/CCL4, MIP-2/CXCL2/GRO/CINC-3, RANTES/CCL5, SDF-1/CXCL12, TARC/CCL17, TIMP-1, TNF-,TREM-1

Rat Cytokine Array, Panel A PRODUCT DESCRIPTION CATALOG # SIZE Rat Cytokine Array Kit, Panel A Contains 4 arrays - each spotted in duplicate with 29 diff erent cytokine antibodies ARY008 1 Kit

CINC-1/CXCL1, CINC-2//CXCL3, CINC-3/CXCL2, CNTF, Fractalkine/CX3CL1, GM-CSF, ICAM-1/CD54, IFN-, IL-1, IL-1/IL-1F2, IL-1ra, IL-2, IL-3, IL-4, IL-6, IL-10, IL-13, IL-17, IP-10/CXCL10, LIX, MIG/CXCL9, MIP-1/CCL3,MIP-3/CCL20,RANTES/CCL5, L-Selectin/CD62L, Thymus Chemokine/CXCL7, TIMP-1, TNF-,VEGF

Mouse Adipokine Array PRODUCT DESCRIPTION CATALOG # SIZE Mouse Adipokine Array Kit Contains 4 membranes - each spotted in duplicate with 38 diff erent obesity-related antibodies ARY013 1 Kit

Adiponectin, AgRP, ANGPT-L3, CRP, DPPIV, Endocan, Fetuin A, FGF acidic, FGF-21, HGF, ICAM-1, IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-5, IGFBP-6, IL-6, IL-10, IL-11, Leptin, LIF, Lipocalin-2/NGAL, MCP-1/JE, M-CSF, Oncostatin M, Pentraxin 2, Pentraxin 3, Pref-1/DLK-1/FA1, RAGE, RANTES, RBP4, Resistin, Serpin E1/PAI-1, TIMP-1, TNF-, VEGF

www.RnDSystems.com/go/Multiplex 87 SPECIALIZED TOOLS

Human Phospho-Immunoreceptor Array PRODUCT DESCRIPTION CATALOG # SIZE Human Phospho-Immunoreceptor Array Kit Contains 4 arrays - each spotted in duplicate with 59 diff erent antibodies recognizing proteins involved ARY004 1 Kit in immunoreceptor signaling.

2B4/SLAMF4, BLAME/SLAMF8, BTLA, CD3,CD5, CD6, CD28, CD84, CD150/SLAM, CD229, CEACAM-1, CLEC-1, CLEC-2, CRACC/SLAMF7, CTLA-4, DCIR, Dectin-1, DNAM-1, Fc RII, Fc RIIA, Fc RIIIA/B, FcRH1, FcRH2, FcRH4, FcRH5, ILT2, ILT3, ILT4, ILT5, ILT6, Integrin 3, KIR2DL4, LAIR-1, LAIR-2, LMIR1, LMIR2, LMIR3, LMIR6, MDL-1, NKp30, NKp44, NKp46, NKp80, NTB-A, PD-1, PECAM, SHIP-1, SHP-1, SHP-2, Siglec-2, Siglec-3, Siglec-5, Siglec-7, Siglec-9, Siglec-10, SIRP-1, TREM-1, TREM-2, TREML1

Human Phospho-Kinase Array PRODUCT DESCRIPTION CATALOG # SIZE Human Phospho-Kinase Array Kit Contains 4 sets of 2 membranes - each spotted in duplicate with 46 diff erent phospho antibodies ARY003 1 Kit

Akt (S473), Akt (T308), AMPK1 (T174), AMPK2 (T172), -Catenin, Chk-2 (T68), CREB (S133), ERK1/2 (T202/Y204, T185/Y187), FAK (Y397), Fgr (Y412), Fyn (Y420), GSK-3/ (S21/S9), Hck (Y411), HSP27 (S78/S82), JNK pan (T183/Y185, T221/Y223), c-Jun (S63), Lck (Y394), Lyn (Y397), MEK1/2 (S218/S222, S222/S226), MSK1/2 (S376/S360), eNOS (S1177), p27 (T198), p27 (T157), p38 (T180/Y182), p53 (S15), p53 (S46), p53 (S392), p70 S6 Kinase (T229), p70 S6 Kinase (T389), p70 S6 Kinase (T421/S424), Paxillin (Y118), PLC-1 (Y783), Pyk2 (Y402), RSK1/2 (S221), RSK1/2/3 (S380), Src (Y419), STAT1 (Y701), STAT2 (Y689), STAT3 (Y705), STAT4 (Y693), STAT5a (Y699), STAT5a/b (Y699), STAT5b (Y699), STAT6 (Y641), TOR (S2448), Yes (Y426)

Human Phospho-Mitogen Activated Protein Kinase (MAPK) Array PRODUCT DESCRIPTION CATALOG # SIZE Human Phospho-MAPK Array Kit Contains 4 arrays - each spotted in duplicate with 26 diff erent antibodies recognizing MAPKs and other ARY002B 1 Kit kinases.

Akt1 (S473), Akt2 (S474), Akt3 (S472), Akt pan (S473, S474, S472), CREB, ERK1 (T202/Y204), ERK2 (T185/Y187), GSK-3/ (S21/S9), GSK-3 (S9), HSP27 (S78/S82), JNK1 (T183/Y185), JNK2 (T183/Y185), JNK3 (T221/Y223), JNK pan (T183/Y185, T221/Y223), MKK3, MKK6, MSK2 (S360), p38 (T180/Y182), p38 (T180/Y182), p38 (T180/Y182), p38 (T183/Y185), p53 (S46), p70 S6 Kinase (T421/S424), RSK1 (S380), RSK2 (S386), TOR

Human Phospho-Receptor Tyrosine Kinase (RTK) Array PRODUCT DESCRIPTION CATALOG # SIZE Human Phospho-RTK Array Kit Contains 4 arrays - each spotted in duplicate with 42 diff erent RTK antibodies ARY001 1 Kit

Axl, Dtk, EGF R, EphA1, EphA2, EphA3, EphA4, EphA6, EphA7, EphB1, EphB2, EphB4, EphB6, ErbB2, ErbB3, ErbB4, FGF R1, FGF R2,FGF R3, FGF R4, Flt-3, HGF R, IGF-I R, Insulin R, M-CSF R, Mer, MSP R, MuSK, PDGF R,PDGF R, c-Ret, ROR1, ROR2, SCF R, Tie-1, Tie-2, TrkA, TrkB, TrkC, VEGF R1, VEGF R2, VEGF R3

Mouse Phospho-Receptor Tyrosine Kinase (RTK) Array PRODUCT DESCRIPTION CATALOG # SIZE Mouse Phospho-RTK Array Kit Contains 4 arrays - each spotted in duplicate with 39 diff erent RTK antibodies ARY014 1 Kit

Axl, Dtk, EGF R, EphA1, EphA2, EphA3, EphA6, EphA7, EphA8, EphB1, EphB2, EphB4, EphB6, ErbB2, ErbB3, ErbB4, FGF R2 (IIIc), FGF R3, FGF R4, Flt-3, HGF R, IGF-I R, Insulin R, M-CSF R, Mer, MSP R, MuSK, PDGF R, PDGF R, c-RET, SCF R, Tie-1, Tie-2, TrkA, TrkB, TrkC, VEGF R1, VEGF R2, VEGF R3

Human Pluripotent Stem Cell Array PRODUCT DESCRIPTION CATALOG # SIZE Human Pluripotent Stem Cell Array Kit Contains 8 arrays - each spotted in duplicate with 15 diff erent stem cell marker antibodies ARY010 1 Kit

E-Cadherin, -Fetoprotein, GATA-4, Goosecoid, HCG, HNF-3/FoxA2, Nanog, Oct-3/4, Otx2, PDX-1/IPF1, Snail, SOX2, SOX17, TP63/TP73L, VEGF R2/KDR/Flk-1

Proteome Proﬁ ler Antibody Arrays: Now for use with LI-COR® Dyes The Proteome Profi ler line of membrane-based planar arrays has now been qualifi ed for use with the near-infrared fl uorescent dyes from LI-COR. Currently, the Human, Mouse, and Rat Proteome Profi ler Cytokine Arrays (Catalog # ARY005, ARY006, ARY008, respectively), and the Human Angiogenesis Array (Catalog # ARY007) can be adapted for use with LI-COR by the simple substitution of IRDye® 800CW Streptavidin (LI-COR Catalog # 926-32230) for the Streptavidin-HRP provided with the kit. The advantages of this substitution include cost savings on chemiluminescence reagents and X-ray fi lm and enhanced dye stability. For LI-COR-adapted protocols and more details about the kits suitable for use with the LI-COR dye, please visit our website at www.RnDSystems.com/go/LI-CORProtocols.

LI-COR and IRDye are registered trademarks of LI-COR Biosciences.

88 For research use only. Not for use in diagnostic procedures. SPECIALIZED TOOLS

Proteome Proﬁ ler 96 The Array in a Well Proteome Profi ler 96 Antibody Arrays consist of a 96-well microplate pre-spotted with a series of capture antibodies in each well. Experimental samples are added and target proteins present in the samples bind to the immobilized antibodies. A cocktail of biotinylated detection antibodies and Streptavidin-HRP or an HRP-conjugated pan anti-Phospho-Tyrosine antibody is subsequently used to detect the bound proteins. Chemiluminescent substrate reagents produce a signal that is proportional to the amount of analyte bound. A camera imaging system capable of chemiluminescent detection is used to determine the intensity of light emitted in individual spots. For more information, please visit our website at www.RnDSystems. com/go/ProteomeProfi ler96.

Human Phospho Receptor Tyrosine Kinase (RTK) Array 1 PRODUCT DESCRIPTION CATALOG # SIZE Proteome Profi ler 96 Contains a 96-well microplate spotted with 16 diff erent RTK antibodies ARZ001 1 Kit Human Phospho-RTK Array 1 Kit

EGFR, ErbB2, ErbB3, ErbB4, HGF R, IGF-I R, Insulin R, M-CSF R, MSP R, PDGF R, PDGF R, SCF R, Tie-2, VEGF R1, VEGF R2, VEGF R3

Human Phospho-Receptor Tyrosine Kinase (RTK) Array 2 PRODUCT DESCRIPTION CATALOG # SIZE Proteome Profi ler 96 Contains a 96-well microplate spotted with 8 diff erent Breast Cancer-related RTK antibodies ARZ002 1 Kit Human Phospho-RTK Array 2 Kit EGF R, EphB4, ErbB2, ErbB3, ErbB4, HGF R, IGF-I R, MSP R

Human Phospho-Receptor Tyrosine Kinase (RTK) Array 3 PRODUCT DESCRIPTION CATALOG # SIZE Proteome Profi ler 96 Contains a 96-well microplate spotted with 8 diff erent Angiogenesis-related RTK antibodies ARZ003 1 Kit Human Phospho-RTK Array 3 Kit

EphB4, PDGF R, PDGF R, Tie-1, Tie-2, VEGF R1, VEGF R2, VEGF R3

Human Phospho-Kinase Array 1

PRODUCT DESCRIPTION CATALOG # SIZE Proteome Profi ler 96 Phospho-Kinase Array 1 Kit Contains a 96-well microplate spotted with 8 diff erent intracellular kinase-related antibodies ARZ004 1 Kit

Akt1 (S473), ERK1/ERK2 (T202/Y204), GSK-3 (S9), JNK Pan (T183/Y185), p38 (T180/Y182), p70 S6 Kinase (T421/S424), Src (Y416)

www.RnDSystems.com/go/Multiplex 89 SPECIALIZED TOOLS

Multiplex Kits for the Luminex® Platform: Fluorokine® MAP R&D Systems off ers a choice of products for Fluorokine Multianalyte Profi ling Multiplex Assays: as a stand-alone pre-mixed kit or as a Base Kit in combination with a panel of Analyte Kits. Each Analyte Kit contains antibody-coated microparticles and biotin-conjugated detection antibodies. The Base Kit contains all of the other reagents needed to perform the assay. This arrangement allows the end-user maximum fl exibility in creating panel composition. For ease of use, we also off er pre-mixed complete kits which contain all the reagents needed to simultaneously determine concentrations of multiple molecules from a single sample. Fluorokine MAP Multiplex Assays are subject to the same development, validation, manufacturing, and quality control standards as our Quantikine® ELISAs, the world’s most referenced immunoassays. The result is accurate, sensitive, and reproducible multiplex assays. For each sample type, the panel is evaluated and validated for sensitivity, precision, recovery, sample linearity, specifi city, and the ability to recognize both the natural and recombinant analyte.* Furthermore, all analytes in a given panel are optimized and tested together to ensure multiplex compatibility. For more information, please visit our website at www.RnDSystems.com/go/Multiplex * For each sample type, a single, multipurpose diluent is used to optimize recovery, linearity and reproducibility. Such a multipurpose diluent may not optimize any single analyte to the same degree that a unique diluent selected for analysis of that analyte can optimize conditions. Therefore, some performance characteristics may be more variable than those for assays designed specifi cally for single analyte analysis.

Human Angiogenesis Panel A BASE KIT CONTENTS CATALOG # Human Angiogenesis Standard Cocktail, Microparticle Diluent, Calibrator Diluent, Wash Buff er Concentrate, Filter-bottomed Microplate, Mixing Bottles, Plate Sealers, LAN000 Panel A Biotin Antibody Diluent, Streptavidin-PE

ANALYTE KIT** SAMPLE TYPE SENSITIVITY*** CATALOG # ANALYTE KIT** SAMPLE TYPE SENSITIVITY*** CATALOG #

Angiogenin C S P U M 2.7 pg/mL LAN265 PDGF-BB C S P U M 2 pg/mL LAN220 Angiopoietin-1 C S P U M 51.1 pg/mL LAN923 PlGF C S P U M 1 pg/mL LAN264 Endostatin C S P U M 31.9 pg/mL LAN1098 Thrombospondin-2 C S P U M 6.71 pg/mL LAN1635 FGF acidic C S P U M 6.7 pg/mL LAN232 VEGF C S P U M 1.84 pg/mL LUH293 FGF basic C S P**** U M 4.91 pg/mL LUH233 VEGF-D C S P U M 19.1 pg/mL LAN622 PDGF-AA C S P U M 1 pg/mL LAN221 ** Analyte Kits are validated only for use with their designated Base Kit. *** Sensitivity values listed here are representative values and can vary by lot. A standard curve must be generated each time an assay is run, utilizing values from the Standard Value Cards included in the Base Kit. **** Not suitable for heparin plasma.

Human MMP Panel BASE KIT CONTENTS CATALOG # Human MMP Panel Standard Cocktail, Microparticle Diluent, Calibrator Diluent, Wash Buff er Concentrate, Filter-bottomed Microplate, Mixing Bottles, Plate Sealers, LMP000 Biotin Antibody Diluent, Streptavidin-PE

ANALYTE KIT** SAMPLE TYPE**** SENSITIVITY*** CATALOG # ANALYTE KIT** SAMPLE TYPE**** SENSITIVITY*** CATALOG #

MMP-1 C S P 6.3 pg/mL LMP901 MMP-8 C S P 12.5 pg/mL LMP908 MMP-2 C S P 38.9 pg/mL LMP902 MMP-9 C S P 11.0 pg/mL LMP911 MMP-3 C S P 2.6 pg/mL LMP513 MMP-12 C S P 1.9 pg/mL LMP919 MMP-7 C S P 29.5 pg/mL LMP907 MMP-13 C S 28.6 pg/mL LMP511 ** Analyte Kits are validated only for use with their designated Base Kit. *** Sensitivity values listed here are representative values and can vary by lot. A standard curve must be generated each time an assay is run, utilizing values from the Standard Value Card included in the Base Kit. **** Not suitable for EDTA & citrate plasma

Sample Type Key: C Cell Culture Supernate S Serum P Plasma U Urine M Human Milk Luminex is a registered trademark of Luminex Corporation

90 For research use only. Not for use in diagnostic procedures. SPECIALIZED TOOLS

Human Obesity Panel BASE KIT CONTENTS CATALOG # Human Obesity Panel Standard Cocktail, Microparticle Diluent, Calibrator Diluents, Wash Buff er Concentrate, Filter-bottomed Microplate, Mixing Bottles, Plate Sealers, LOB000 Biotin Antibody Diluent, Streptavidin-PE

ANALYTE KIT** SAMPLE TYPE SENSITIVITY*** CATALOG # ANALYTE KIT** SAMPLE TYPE SENSITIVITY*** CATALOG #

Adiponectin/Acrp30 C S P 19.8 pg/mL. LOB1065 IL-10 C S P 0.30 pg/mL LUH217 C-Reactive Protein C S P 1.9 pg/mL LOB1707 Leptin C S P 20.2 pg/mL LUB398 CCL2/MCP-1 C S P 0.47 pg/mL LUH279 Resistin C S P 7.3 pg/mL LOB1359 Complement Factor C S P 3.5 pg/mL LOB1824 Serpin E1/PAI-1 C S P 0.29 pg/mL LOB1786 D/Adipsin TNF- C S P 1.50 pg/mL LUH210 IL-6 C S P 1.11 pg/mL LUH206 ** Analyte Kits are validated only for use with their designated Base Kit. *** Sensitivity values listed here are representative values and can vary by lot. A standard curve must be generated each time an assay is run, utilizing values from the Standard Value Card included in the Base Kit.

Human Cytokine Panel A BASE KIT CONTENTS CATALOG # Human Cytokine Standard Cocktails, Microparticle Diluent, Calibrator Diluents, Wash Buff er Concentrate, Filter-bottomed Microplate, Mixing Bottles, Plate Sealers, LUH000 Panel A Biotin Antibody Diluent, Streptavidin-PE

ANALYTE KIT** SAMPLE TYPE SENSITIVITY*** CATALOG # ANALYTE KIT** SAMPLE TYPE SENSITIVITY*** CATALOG #

CCL2/MCP-1 C S P 0.47 pg/mL LUH279 IL-1/IL-1F2 C S P 0.57 pg/mL LUH201 CCL3/MIP-1 C S P 1.45 pg/mL LUH270 IL-1ra/IL-1F3 C S P 10.91 pg/mL LUH280 CCL4/MIP-1 C S P 0.72 pg/mL LUH271 IL-2 C S P 2.23 pg/mL LUH202 CCL5/RANTES C S P 1.91 pg/mL LUH278 IL-4 C S P 4.46 pg/mL LUH204 CXCL5/ENA-78 C S P 4.14 pg/mL LUH254 IL-5 C S P 0.71 pg/mL LUH205 CXCL8/IL-8 C S P 1.97 pg/mL LUH208 IL-6 C S P 1.11 pg/mL LUH206 FGF basic C S P**** 4.91 pg/mL LUH233 IL-10 C S P 0.30 pg/mL LUH217 G-CSF C S P 1.48 pg/mL LUH214 IL-17 C S P 1.10 pg/mL LUH317

GM-CSF C S P 1.98 pg/mL LUH215C TNF- C S P 1.50 pg/mL LUH210 IFN- C S P 1.27 pg/mL LUH285 Tpo C S P 9.94 pg/mL LUH288 IL-1/IL-1F1 C S P 0.36 pg/mL LUH200B VEGF C S P 1.84 pg/mL LUH293 ** Analyte Kits are validated only for use with their designated Base Kit. Human Panel A controls are also available, please inquire. *** Sensitivity values listed here are representative values and can vary by lot. A standard curve must be generated each time an assay is run, utilizing values from the Standard Value Cards included in the Base Kit. **** Not suitable for heparin plasma

Human Cytokine Panel B BASE KIT CONTENTS CATALOG # Human Cytokine Standard Cocktail, Microparticle Diluent, Calibrator Diluents, Wash Buff er Concentrate, Filter-bottomed Microplate, Mixing Bottles, Plate Sealers, LUB000 Panel B Biotin Antibody Diluent, Streptavidin-PE

ANALYTE KIT** SAMPLE TYPE SENSITIVITY*** CATALOG # ANALYTE KIT** SAMPLE TYPE SENSITIVITY*** CATALOG #

CCL11/Eotaxin C S P 8.2 pg/mL LUB320 HGF C S P 4.1 pg/mL LUB294 CD40 Ligand/TNFSF5 C S P 23.4 pg/mL LUB201 IL-12 p70 C S P 14.9 pg/mL LUB219 CXCL10/IP-10 C P 0.10 pg/mL LUB266 IL-13 C S P 15.6 pg/mL LUB213 CXCL11/I-TAC C S P 22.5 pg/mL LUB672 Leptin C S P 20.2 pg/mL LUB398 EGF C S P 1.58 pg/mL LUB236 ** Analyte Kits are validated only for use with their designated Base Kit. *** Sensitivity values listed here are representative values and can vary by lot. A standard curve must be generated each time an assay is run, utilizing values from the Standard Value Card included in the Base Kit.

www.RnDSystems.com/go/Multiplex 91 SPECIALIZED TOOLS

Mouse Cytokine Panel BASE KIT CONTENTS CATALOG # Mouse Cytokine Panel Standard Cocktail, Microparticle Diluent, Calibrator Diluents, Wash Buff er Concentrate, Filter-bottomed Microplate, Mixing Bottles, Plate Sealers, Biotin LUM000 Antibody Diluent, Streptavidin-PE

ANALYTE KIT** SAMPLE TYPE SENSITIVITY*** CATALOG # ANALYTE KIT** SAMPLE TYPE SENSITIVITY*** CATALOG #

CCL2/JE/MCP-1 C S P 37.1 pg/mL LUM479 IL-5 C S P 4.18 pg/mL LUM405 CXCL1/KC C S P 2.69 pg/mL LUM453 IL-6 C S P 1.06 pg/mL LUM406 CXCL2/MIP-2 C S P 10.1 pg/mL LUM452 IL-10 C S P 1.28 pg/mL LUM417

GM-CSF C S P 3.29 pg/mL LUM415B IL-12 p70 C S P 22.42 pg/mL LUM419

IFN- C S P 7.6 pg/mL LUM485 IL-13 C S P 42.0 pg/mL LUM413

IL-1/IL-1F2 C S P 7.82 pg/mL LUM401 IL-17 C S P 10.8 pg/mL LUM421

IL-2 C S P 6.3 pg/mL LUM402 TNF- C S P 1.39 pg/mL LUM410 IL-4 C 4.27 pg/mL LUM404 VEGF C S P 10.9 pg/mL LUM493 ** Analyte Kits are validated only for use with their designated Base Kit. Mouse Panel controls are also available, please inquire. *** Sensitivity values listed here are representative values and can vary by lot. A standard curve must be generated each time an assay is run, utilizing values from the Standard Value Card included in the Base Kit.

Rat Cytokine Panel BASE KIT CONTENTS CATALOG # Rat Cytokine Panel Standard Cocktail, Microparticle Diluent, Calibrator Diluents, Wash Buff er Concentrate, Filter-bottomed Microplate, Mixing Bottles, Plate Sealers, Biotin LUR000 Antibody Diluent, Streptavidin-PE

ANALYTE KIT** SAMPLE TYPE SENSITIVITY*** CATALOG # ANALYTE KIT** SAMPLE TYPE SENSITIVITY*** CATALOG #

CXCL2/CINC-3 C S P 8.44 pg/mL LUR525 IL-6 C S P 71.3 pg/mL LUR506

CXCL3/CINC-2/ C S P 3.59 pg/mL LUR516 IL-10 C S P 11.6 pg/mL LUR522 GM-CSF C S P 3.13 pg/mL LUR518 IL-13 C S P 6.99 pg/mL LUR1945 ICAM-1/CD54 C S P 3.40 pg/mL LUR583 IL-18/IL-1F4 C S P 10.8 pg/mL LUR521

IFN- C S P 137 pg/mL LUR585 L-Selectin/CD62L C S P 51.9 pg/mL LUR1534 IL-1/IL-1F1 C S P 20.9 pg/mL LUR500 TIMP-1 C S P 6.5 pg/mL LUR580 IL-1/IL-1F2 C S P 16.6 pg/mL LUR501 TNF- C S P 19.5 pg/mL LUR510 IL-2 C S P 8.72 pg/mL LUR502 VEGF C S P 3.51 pg/mL LUR564 IL-4 C S P 1.84 pg/mL LUR504 ** Analyte Kits are validated only for use with their designated Base Kit. *** Sensitivity values listed here are representative values and can vary by lot. A standard curve must be generated each time an assay is run, utilizing values from the Standard Value Card included in the Base Kit.

Pre-mixed Fluorokine® MAP Multiplex Kits Pre-mixed Kits contain analyte-specifi c capture antibodies.

Human Inﬂ ammation 12-Plex Kit PRODUCT DESCRIPTION CATALOG # SIZE Human Infl ammation For the Measurement of CXCL8/IL-8, GM-CSF, IFN-IL-1/IL-1F2IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-, andVEGF in a single LKT005 1 Kit 12-Plex Kit sample

ANALYTE SAMPLE TYPE SENSITIVITY*** ANALYTE SAMPLE TYPE SENSITIVITY***

CXCL8/IL-8 Serum, Plasma (EDTA & Heparin) 0.067 pg/mL IL-6 Serum, Plasma (EDTA & Heparin) 0.306 pg/mL

GM-CSF Serum, Plasma (EDTA & Heparin) 0.128 pg/mL IL-10 Serum, Plasma (EDTA & Heparin) 0.235 pg/mL

IFN- Serum, Plasma (EDTA & Heparin) 0.078 pg/mL IL-12 Serum, Plasma (EDTA & Heparin) 2.955 pg/mL IL-1 /IL-1F2 Serum, Plasma (EDTA & Heparin) 0.179 pg/mL  TNF- Serum, Plasma (EDTA & Heparin) 0.536 pg/mL IL-2 Serum, Plasma (EDTA & Heparin) 0.283 pg/mL VEGF Serum, Plasma (EDTA & Heparin) 1.346 pg/mL IL-4 Serum, Plasma (EDTA & Heparin) 2.539 pg/mL IL-5 Serum, Plasma (EDTA & Heparin) 0.115 pg/mL

*** Sensitivity values listed here are representative values and can vary by lot. A standard curve must be generated each time an assay is run, utilizing values from the Standard Value Card included in the kit.

Sample Type Key: C Cell Culture Supernate S Serum P Plasma U Urine M Human Milk

92 For research use only. Not for use in diagnostic procedures. SPECIALIZED TOOLS

Two New Methods for Creating Custom Luminex®-based Panels R&D Systems now off ers researchers two new methods for creating custom analyte panels for Luminex-based multianalyte profi ling assays. The Custom Premix Fluorokine Multianalyte Profi ling (MAP) Kits and the Human VersaMAP™ Multiplex Development System give researchers the opportunity to specify their own unique panel of analytes for bead-based multianalyte profi ling.

Custom Premix Fluorokine MAP Kits VersaMAP Multiplex Development System Custom Premix FMAP Kits allow researchers to select up to 10 analytes The Human VersaMAP Development System allows the selection from one of our existing multiplex panels (listed below). Capture and of up to 10 analytes from a large and continuously expanding list detection reagents for these analytes are provided as premixed cocktails, of potential analytes. VersaMAP off ers the widest selection and and can be used to assay cell culture supernates, serum, or plasma greatest fl exibility for creating your panel of choice. Reagents for samples. A specialized on-line ordering tool has been created to make the VersaMAP Kits are validated for use with cell culture supernates, customizing your own Fluorokine MAP panel easier than ever. This tool and may be used with more complex sample types following further allows you to select the species of interest (Step 1), your panel of choice optimization by the user. An on-line ordering tool has been designed (Step 2), and the analytes in that panel that you want to be included in to guide users through the ordering process. To help decide which your custom kit (Step 3). custom format is right for you, consult our comparison table at www.RnDSystems.com/go/LuminexComparison.

Fluorokine MAP Custom Premix Configuration & Ordering Tool VersaMAP Development System Configuration & Ordering Tool Retrieve a Saved Configuration > Configure your custom VersaMAP Development System by selecting up to 10 analytes. STEP Select a Species You may save your configuration to retrieve it at a later time. 1 ORDERORDER SUMMARYSUMMARY Human A 3-5 day turn-around time is required to process orders. Pleasese aallowllow addadditionalitional ttimeime for internatinternationalionaliona ororders.ders. All VersVersaMAPaMAP ororders are final and may not be canceled following order confirmation.. Mouse CatalogCatalotlg # FCST02FCST02-04-040 Rat Help me design my custom premix panel AnalytesAnalytes SelSelected:ected: Retrieve a Saved Configuration > STEP Select a Panel 1. Angiopoietin-1 SELECT UP TO 10 ANALYTES 2 Human Angiogeneis Custom Premix Fluorokine MAP Kit, 1 Plate 2. FGF basic Adiponectin/Acrp30 IL-17A ORDERER SUMMARYSUMMAMARY CCL2/MCP-1 IL-1ra 3. PlGF CCL3/MIP-1 alpha IL-2 Catalog # CCL4/MIP-1 beta IL-4 STEP Select from the available analytes listed below 4. VEGF Price:Price: CCL5/RANTES IL-5 Analytes Selected: 3 Complement Factor D/Adipsin IL-6 CRP Leptin CXCL11/I-TAC MMP-1 Ord ns an ns & CXCL5/ENA-78 MMP-13 ne er tio d io re li in c st ct tu SELECT UP TO 10 ANALYTES n g le a e r r l n O e t e SAVE & s s CLEAR l CXCL8/IL-8 MMP-2 o

r RETURN v e

SELECTIONS t a

l a r

E-Selectin MMP-3 s c

Angiogenin PDGF-BB

Ŀ Ŀ FGF basic MMP-7 Angiopoietin-1 PlGF Ord ns an s & Ŀ Ŀ ne er tio d ion re G-CSF MMP-8 ADD TO CART RESET SAVE/ORDER li in c st ct tu n g le a e r r l n GM-CSF MMP-9 Endostatin Thrombospondin-2 O e t e SAVE & Ŀ Ŀ s CLEAR s l o a

r RETURN v

SELECTIONS e t ICAM-1 P-Selectin a e

FGF acidic VEGF r

l a

Ŀ Ŀ

c IFN-gamma Thrombopoietin/Tpo

Ŀ FGF basic Ŀ VEGF-D IL-1 alpha TNF-alpha IL-1 beta VCAM-1 PDGF-AA ADD TO CART RESET SAVE Ŀ IL-10 VEGF IL-12 p70

Available Analytes for the Custom Premix Fluorokine MAP Kits: Available Analytes for VersaMAP Development Systems: Select up to 10 analytes from one of the panels listed below. Select up to 10 analytes from the list below. Human Angiogenesis Panel A Catalog # FCST02 Adiponectin/Acrp30 ICAM-1/CD54 MMP-1 Angiogenin, Angiopoietin-1, Endostatin, FGF acidic, FGF basic, PDGF-AA, PDGF-BB, PlGF, Thrombospondin-2, VEGF, VEGF-D CCL2/MCP-1 IFN- MMP-2 Human Cardiac Panel A Catalog # FCST11 CCL3/MIP-1 IL-1/IL-1F1 MMP-3 CD40 Ligand, GDF-15, Pappalysin/PAPP-A, PCSK9, ST2/IL-1 R4, TNF RII CCL4/MIP-1 IL-1/IL-1F2 MMP-7 CCL5/RANTES IL-1ra/IL-1F3 MMP-8 Human Cytokine Panel A Catalog # FCST03 Complement Factor D IL-2 MMP-9 CCL2/MCP-1, CCL3/MIP-1, CCL4/MIP-1, CCL5/RANTES, CXCL5/ENA-78, CXCL8/IL-8, FGF basic, G-CSF, GM-CSF, IFN-, IL-1/IL-1F1, IL-1/IL-1F2, IL-1ra/IL-1F3, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, TNF-, C-Reactive Protein IL-4 MMP-13 Tpo, VEGF CXCL11/I-TAC IL-5 E-Selectin Human Cytokine Panel B Catalog # FCST04 CXCL5/ENA-78 IL-6 P-Selectin CCL11/Eotaxin, CD40 Ligand/TNFSF5, CXCL10/IP-10, CXCL11/I-TAC, EGF, HGF, IL-12 p70, IL-13, Leptin CXCL8/IL-8 IL-10 TNF- Human Obesity Panel Catalog # FCST08 FGF basic IL-12 p70 Tpo Adiponectin/Acrp30, C-Reactive Protein, CCL2/MCP-1, Complement Factor D, IL-6, IL-10, Leptin, G-CSF IL-17A VCAM-1 Resistin, Serpin E1/PAI-1, TNF- Mouse Cytokine Panel Catalog # FCST05 GM-CSF Leptin VEGF CCL2/JE, CXCL1/KC, CXCL2/MIP-2, GM-CSF, IFN-, IL-1/IL-1F2, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p70, IL-13, IL-17, TNF-, VEGF Rat Cytokine Panel Catalog # FCST06 For more information on the Custom Fluorokine MAP Kits or CXCL2/CINC-3, CXCL3/CINC-2/, GM-CSF, ICAM-1/CD54, IFN-, IL-1/IL-1F1, IL-1/IL-1F2, IL-2, IL-4, VersaMAP Development System, please visit our website at IL-6, IL-10, IL-13, IL-18/IL-1F4, L-Selectin/CD62L, TIMP-1, TNF-, VEGF www.RnDSystems.com/go/LuminexComparison Fluorokine is a registered trademark of R&D Systems. Luminex is a registered trademark of Luminex Corporation www.RnDSystems.com/go/LuminexComparison 93 SPECIALIZED TOOLS

Mosaic™ ELISA: A New Quantitative Microplate-based Multiplex Technology Mosaic ELISA Kits are a rapid, convenient, microplate-based multiplex system that simultaneously quantitates up to 8 analytes in a single well. Every well is pre-spotted with antibodies specifi c for each analyte of interest. A chemiluminescent substrate, detection antibody cocktail, and all buff ers are included in the kit. A chemiluminescence camera imaging system, such as the Q-View™ Imager by Quansys Biosciences, is used to photograph the microplate. Each spot is analyzed for pixel intensity. The intensity of each spot in the well is directly proportional to the amount of analyte present in each sample. Please see our website at www.RnDSystems.com/go/Mosaic for additional suitable imaging systems.

Human Cytokine Panel 1 PRODUCT CATALOG # SIZE Mosaic ELISA Human Cytokine Panel 1 Kit MEA001 1 Kit

ANALYTE SAMPLE TYPE SENSITIVITY ANALYTE SAMPLE TYPE SENSITIVITY

CXCL8/IL-8 S P C 1.24 pg/mL IL-1/IL-1F2 S P C 0.31 pg/mL CD40 Ligand S P C 5.31 pg/mL IL-6 S P C 0.76 pg/mL

IFN- S P C 2.08 pg/mL IL-17 S P C 0.35 pg/mL IL-1/IL-1F1 S P C 0.74 pg/mL TNF- S P C 2.13 pg/mL

Human Growth Factor Panel 1 PRODUCT CATALOG # SIZE Mosaic Human Growth Factor Panel 1 Kit MEA004 1 Kit

ANALYTE SAMPLE TYPE SENSITIVITY ANALYTE SAMPLE TYPE SENSITIVITY

FGF basic S P C 2.55 pg/mL PDGF-BB S P C 6.72 pg/mL G-CSF S P C 13.25 pg/mL PlGF S P C 2.64 pg/mL HGF S P C 9.33 pg/mL VEGF S P C 7.73 pg/mL

NEURAL STEM CELL CULTURE MEDIA & KITS Pluripotent Stem Cells: Feeder Cells, Media, Markers Pluripotent stem cells, which are derived from the inner cell mass of preimplantation embryos or induced by expression of a set of stem-cell related genes, have been recognized as the most pluripotent stem cell population. These cells are capable of unlimited, undiff erentiated proliferation in vitro and still maintain the capacity for diff erentiation into a wide variety of somatic tissues. In this capacity pluripotent stem cells have widespread clinical potential in the treatments of heart disease, diabetes, spinal cord injury, and a variety of neurodegenerative disorders. R&D Systems off ers a range of products designed to maintain and expand pluripotent stem cells in culture, and to monitor their diff erentiation status.

Stem/Progenitor Cell Growth Substrate StemXVivo™ Culture Matrix is a fully defi ned, proprietary mixture of recombinant adhesion molecules designed for the culture of stem/progenitor cells. It can be used as a substitute for Engelbreth-Holm-Swarm (EHS) basement membrane extract or a feeder layer in the maintenance and/or diff erentiation of stem/progenitor cells. The culture matrix is tested for its ability to support the attachment and growth of multiple stem/progenitor cell populations.

PRODUCT DESCRIPTION CATALOG # SIZE StemXVivo Culture Matrix (100X) A defi ned proprietary mixture of recombinant human adhesion molecules for the culture of CCM013 1 mL A defi ned substitute for EHS extracts stem/progenitor cells.

Pluripotent Cell-derived Endoderm Diﬀ erentiation Kit R&D Systems off ers a kit for the diff erentiation of human pluripotent stem cells into defi nitive endoderm. The kit contains specially formulated media supplements and growth factors that can be used to direct the serum-free diff erentiation of 2.8 x 107 pluripotent cells into defi nitive endoderm.

PRODUCT DESCRIPTION CATALOG # SIZE Pluripotent Cell-derived Endoderm Diff er- Media supplement and growth factors for the diff erentiation of human pluripotent stem cells into SC019 250 mL entiation Kit (Includes media supplements defi nitive endoderm. & growth factors)

Sample Type Key: C Cell Culture Supernate S Serum P Plasma U Urine M Human Milk

94 For research use only. Not for use in diagnostic procedures. SPECIALIZED TOOLS

Feeder Cells Cryopreserved irradiated mouse embryonic fi broblasts (iMEFs) are isolated from pathogen-free E13.5 CF1 embryos and irradiated at passage 3 to arrest mitosis. One order includes 5 vials, containing approximately 6 x 106 cells/vial. Each lot of iMEF feeders is tested for its ability to maintain the expression levels of pluripotency markers as evaluated by immunocytochemistry or fl ow cytometry.

PRODUCT DESCRIPTION CATALOG # SIZE Irradiated Mouse Embryonic Fibroblasts Tested for their ability to support the expansion of the BG01V human embryonic stem cells in the PSC001 5 Vials undiff erentiated state based on the expression of Oct-3/4 and SSEA-4.

Feeder Cell Conditioned Media Human pluripotent stem cells can be maintained and expanded using mouse or human feeder cell conditioned media. R&D Systems off ers both Mouse Embryonic Fibroblast Conditioned Media and Human Feeder Cell Conditioned Media capable of supporting the growth of cells expressing markers of pluripotency.

PRODUCT DESCRIPTION CATALOG # SIZE Mouse Embryonic Fibroblast Conditioned Serum-free media tested for its ability to support Oct-3/4+, SSEA-4+ growth of the BG01V human AR005 100 mL Media embryonic stem cells in vitro.

Pluripotent Stem Cell 3-Color Immunocytochemistry Kit R&D Systems off ers both antibody- and primer-based kits for the identifi cation of human pluripotent stem cells. The Human Pluripotent Stem Cell 3-Color Immunocytochemistry Kit provides three fl uorochrome-conjugated antibodies for single-step staining of human pluripotent stem cells by immunocytochemistry. The antibodies provided recognize three diff erent markers of pluripotency.

PRODUCT DESCRIPTION CATALOG # SIZE Pluripotent Stem Cell 3-Color NL557-conjugated Anti-SOX2, NL637-conjugated Anti-Oct-3/4, and NL493-conjugated Anti-Nanog SC021 1 Kit Immunocytochemistry Kit

Embryonic Stem Cell Marker Panels Kits for assessing the diff erentiation status of human embryonic stem cells and other pluripotent stem cells. Multiple kits are off ered using either antibody or primer-based identifi cation techniques. These kits provide an economical alternative to buying separate reagents.

PRODUCT DESCRIPTION CATALOG # SIZE Human Embryonic Stem Cell Marker Anti-alkaline phosphatase, Anti-Nanog, Anti-Oct-3/4, Anti-SSEA-1, Anti-SSEA-4 SC008 1 Kit Antibody Panel Human Embryonic Stem Cell Marker Anti-E-Cadherin, Anti-CD9, Anti-Nanog, Anti-Oct-3/4, Anti-Podocalyxin, Anti-SOX2, Anti-SSEA-1, SC009 1 Kit Antibody Panel Plus Anti-SSEA-4

Human Pluripotent Stem Cell AFP, Brachyury, DPPA5/ESG1, GAPDH, GATA-4, HNF-3, Nanog, Nestin, Oct-3/4, Otx2, PDX1, SOX2, SOX17, SC012 1 Kit Assessment Primer Pair Panel TP63/TP73L, and Stella

Mouse/Rat Pluripotent Stem Cell AFP, Brachyury, DPPA5/ESG1, GAPDH, GATA-4, HNF-3, Nanog, Nestin, Oct-3/4, Otx2, PDX1, SOX2, SOX17, SC015 1Kit Assessment Primer Pair Panel TP63/TP73L, and Stella

www.RnDSystems.com/go/StemCells 95 SPECIALIZED TOOLS

Neural Stem Cells: Expansion, Diﬀ erentiation, Markers, Primary Cells Neural stem cells (NSCs) are functionally defi ned by their capacity to self renew and their ability to diff erentiate into multiple specialized progenitor cells that can commit to further maturation along specifi c lineages, including astrocytes, neurons, and oligodendrocytes. Ex vivo expanded neural stem cells serve as excellent tools for researchers studying neural development and neurological disorders. R&D Systems off ers media and NSC expansion kits and functional identifi cation kits to direct stem cells along neural lineages.

N-2 MAX Media Supplement A serum-free, chemically defi ned, concentrated media supplement formulated to provide optimal growth conditions for neural stem cell expansion. N-2 MAX is composed of Human Insulin, Human Transferrin, Putrescine, Selenite, and Progesterone. The supplement is supplied as a 100X concentrate in water.

PRODUCT DESCRIPTION CATALOG # SIZE N-2 MAX Media Supplement A modifi cation of Bottenstein’s formulation, providing optimal conditions for neural stem cell expansion. AR009 5 mL Suffi cient for 500 mL of medium.

Neural Diﬀ erentiation Kits Kits are available for the serum-free diff erentiation of embryonic stem (ES) cells into dopaminergic neurons or oligodendrocytes. Each kit contains specially formulated media supplements and a growth factor panel designed to direct the diff erentiation to the specifi c neural lineage. These kits contain suffi cient reagents for the induction of approximately 3 x 107 ES cells.

PRODUCT KIT CONTENTS CATALOG # SIZE Human/Mouse Dopaminergic Neuron Dif- ITS Media Supplement, N-2 Plus Media Supplement, FGF basic, FGF-8b, Fibronectin, Shh-N SC001B 1 Kit ferentiation Kit Mouse Oligodendrocyte Diff erentiation Kit ITS Media Supplement, N-2 Plus Media Supplement, EGF, FGF basic, Fibronectin, PDGF-AA SC004 1 Kit

Neural Multi-Color Immunocytochemistry Kit Kit contains three fl uorochrome-conjugated primary antibodies that can be used together for single-step ICC staining of human, mouse, or rat neural cell types. The antibodies included in the kit specifi cally stain oligodendrocytes, neurons, or astrocytes to simplify the identifi cation of these three cell types.

PRODUCT KIT CONTENTS CATALOG # SIZE Human/Mouse/Rat Neural 3-Color NL557-conjugated Anti-Oligodendrocyte Marker O4, NL637-conjugated Anti--III Tubulin, SC024 1 Kit Immunocytochemistry Kit NL493-conjugated Anti-GFAP

Neural Precursor Cell-based Screening & Bioassay Kit The Neural Precursor Cell-based Screening & Bioassay Kit is intended as an in vitro screening tool to determine how bioactive agents, such as toxins, drugs, and growth factors, infl uence neural precursor diff erentiation and proliferation.

PRODUCT KIT CONTENTS CATALOG # SIZE Neural Precursor Cell-based Screening Neural Stem Cell Maintenance Supplement, Neural Diff erentiation Supplement, Fibronectin, SC014 1 Kit & Bioassay Kit HRP-conjugated Anti-Neuron-Specifi c -III Tubulin, Resazurin, Buff ers, Substrates, and Diluents

Neural Progenitor Cell Marker Kit The Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel is designed for the identifi cation and characterization of human, mouse, or rat neural progenitor cells by marker expression using immunocytochemistry and fl ow cytometry techniques.

PRODUCT KIT CONTENTS CATALOG # SIZE Human/Mouse/Rat Neural Progenitor Cell Contains 25 g each of antibodies to rat Notch-2, human CXCR4, human Vimentin, human/mouse SC025 1 Kit Marker Antibody Panel SSEA-1, human Musashi-1, human SOX1, human/mouse SOX2, and rat Nestin

96 For research use only. Not for use in diagnostic procedures. SPECIALIZED TOOLS

Primary Cortical Stem Cells R&D Systems off ers ready-to-use primary cortical stem cells isolated from E14.5 Sprague-Dawley rats or E14.5 CD-1 mice. Every lot is tested for high levels of Nestin expression and the capacity for multi-lineage diff erentiation (astrocytes, neurons, and oligodendrocytes). Depending on your research needs, cortical stem cells can be optimally expanded as monolayers or neurospheres.

PRODUCT KIT CONTENTS CATALOG # SIZE Rat Cortical Stem Cells 1 vial; Vial contains 3 x 106 cells NSC001 1 vial

Mouse Cortical Stem Cells 2 vials; Each vial contains 2 x 106 cells NSC002 2 vials

NEURONAL CELL CULTURE PRODUCTS Mycoplasma Detection Kit Mycoplasma contamination alters the phenotypic characteristics of eukaryotic cells in culture and can negatively impact results. R&D Systems microplate-based MycoProbe® Mycoplasma Detection Kit represents a breakthrough for sensitive and reliable routine screening of cell cultures for mycoplasma contamination. This assay detects the eight mycoplasma species known to cause 95% of mycoplasma contamination in cell cultures by measuring mycoplasma 16S ribosomal RNA in lysed cell pellet or cell culture supernate samples.

PRODUCT KIT CONTENTS CATALOG # SIZE MycoProbe Mycoplasma Detection Kit Cell Lysis Buff er, Conjugate, Substrate/Amplifi er Reagents, Diluents, Hybridization Plate, CUL001B 1 Kit* Streptavidin-coated Plate, Pan-specifi c Oligonucleotide Probes, Capture & Detection Probes, Positive Control, Package Insert *Each kit is suffi cient to run up to 96 tests.

Extracellular Matrix: Coated Plates, Basement Membrane Extracts, Invasion Assays Extracellular matrix (ECM) is made up of proteins that include collagen, fi bronectin, laminin, and vitronectin. These molecules interact with cells via integrins and other cell surface receptors, facilitating the adhesion of cells to the ECM. The resulting focal adhesions or focal contacts are important for the maintenance of tissue architecture and for supporting a variety of cellular processes. ECM protein/integrin binding initiates a complex network of signal transduction cascades that, depending on the context, play an important role in cell spreading, migration, proliferation, and diff erentiation during embryogenesis, wound healing, and tumor development.

Extracellular Matrix Protein Pre-coated Plates for Adhesion Assays PRODUCT SPECIES CATALOG # SIZE* Fibronectin Coated 96-well microplates Bovine CWP002 1 Pack Fibronectin Coated 96-well microplates Human CWP001 1 Pack Vitronectin Coated 96-well microplates Bovine CWP004 1 Pack

Vitronectin Coated 96-well microplates Human CWP003 1 Pack *Each pack contains 5 microplates.

www.RnDSystems.com/go/StemCells 97 SPECIALIZED TOOLS

Basement Membrane Extracts

Cultrex® Basement Membrane Extract (BME) is a soluble basement membrane extract of the Engelbreth-Holm-Swarm (EHS) tumor that gels at 37 °C to form a reconstituted basement membrane. It consists of laminin I, type IV collagen, entactin, and heparan sulfate proteoglycan. Cultrex BME is available with or without phenol red and in regular or reduced growth factor formulations. Rigorous Quality Control Standards Ensure: • High Concentration - supports in vivo angiogenesis and tumorigenicity assays • Cell Culture Qualifi ed - no bacterial or fungal growth after 14 days at 37 °C, no mycoplasma contamination, and endotoxin levels < 20 EU/mL • Gel Formation Verifi ed - gels quickly and maintains this form with culture medium for a period of 14 days at 37 °C • In Vitro Assays - promotes diff erentiation of endothelial cells into capillary-like structures and supports angiogenesis in a rat aortic ring assay

PRODUCT PHENOL RED CATALOG # SIZE Cultrex 3D Culture Matrix Basement Membrane Extract Reduced Growth Factor Absent 3445-005-01 5 mL Cultrex 3D Culture Matrix Rat Collagen I Absent 3447-020-01 100 mg Cultrex 3D Culture Matrix Laminin I Absent 3446-005-01 5 mL Cultrex Basement Membrane Extract Absent 3432-005-01 5 mL Cultrex Basement Membrane Extract - Phenol Red Present 3430-005-01 5 mL Cultrex Basement Membrane Extract Reduced Growth Factor Absent 3433-005-01 5 mL Cultrex Basement Membrane Extract Reduced Growth Factor Present 3431-005-01 5 mL Cultrex PathClear® BME Absent 3432-005-02 5 mL Cultrex PathClear Human BME Absent 3415-001-02 1 mL Cultrex PathClear BME - Phenol Red Present 3430-005-02 5 mL Cultrex PathClear BME Reduced Growth Factor Absent 3433-005-02 5 mL Cultrex PathClear BME Reduced Growth Factor - Phenol Red Present 3431-005-02 5 mL Cultrex High Protein Concentration Basement Membrane Extract (HC20+T) PathClear Absent 3444-005-02 5 mL

Additional ECM Related Reagents PRODUCT DESCRIPTION CATALOG # SIZE Cultrex Cell Staining Kit The Cell Staining Kit provides a ready-to-use staining solution, CS Solution, and Wash Solution. The CS 3437-100-K 1 Kit Solution contains a mixture of Azur A and Methylene Blue specially formulated to provide optimized staining of cells and structures grown on Basement Membrane Extract (BME). Cultrex Poly-L-Lysine Poly-L-Lysine, a highly positively charged amino acid chain, is commonly used as a coating agent to 3438-100-01 100 mL promote cell adhesion in culture. This solution is provided ready to use at 0.01% and contains polymers in the 70,000 - 150,000 kDa range.

Cultrex and PathClear are registered trademarks of Trevigen, Inc. 3-D Culture Matrix is a trademark of Trevigen, Inc.

98 For research use only. Not for use in diagnostic procedures. SPECIALIZED TOOLS

Flow Cytometry, Western Blotting, & Cell/Tissue Staining Multi-Color Flow Cytometry Kits* PRODUCT DESCRIPTION CATALOG # SIZE Human Multipotent Mesenchymal Contains conjugated antibodies to human CD105-PerCP (Clone 166707), CD146-Fluorescein (Clone 128018), FMC002 25 Tests Stromal Cell 4-Color Flow Kit CD90-APC (CloneThy-1A1), and CD45-PE (Clone ICRF 2D1) Human Th1 Cell 4-Color Contains conjugated antibodies to human CD3-PerCP (Clone UCHT1), T-bet-Fluorescein (Clone 525803), FMC009 25 Tests Flow Cytometry Kit IL-12 R2-APC (Clone 305719), and IFN--PE (Clone 25723) Human Th2 Cell 4-Color Contains conjugated antibodies to human STAT6-APC, IL-4-Fluorescein, IL-4 R-PE, CD3-PerCP FMC011 25 Tests Flow Cytometry Kit Human Th17 Cell 4-Color Contains conjugated antibodies to human IL-17-PerCP (Clone 41802), CD3-Fluorescein (Clone UCHT1), FMC007 25 Tests Flow Cytometry Kit IL-22-APC (Clone 142928), and IL-23 R-PE (Clone 218213) Human Treg 3-Color Contains conjugated antibodies to human FoxP3-APC, CD25-PE, CD4-PerCP FMC013 50 Tests Flow Cytometry Kit Human/Mouse Embryonic Stem Cell Contains conjugated antibodies to SSEA-1-PerCP (Clone MC-480), SSEA-4-Fluorescein (Clone MC-813-70), FMC001 25 Tests 4-Color Flow Cytometry Kit Oct-3/4-APC (Clone 240408), Sox2-PE (Clone 245610) Mouse Hematopoietic Progenitor Cell Contains conjugated antibodies to mouse CD244-Fluorescein (Goat IgG), CD150-APC (Clone 459911), FMC005 25 Tests 3-Color Flow Kit CD48-PE (Clone 331504) Mouse Th1 Cell 4-Color Contains conjugated antibodies to mouse IL-12 Rβ2-APC, IFN-γ-Fluorescein, CD4-PE, T-bet-PerCP FMC010 25 Tests Flow Cytometry Kit Mouse Th2 Cell 4-Color Contains conjugated antibodies to mouse STAT6-APC, IL-4 R-Fluorescein, IL-5-PE, CD4-PerCP FMC012 25 Tests Flow Cytometry Kit Mouse Th17 Cell 4-Color Contains conjugated antibodies to mouse CD4-APC, CCR6-Fluorescein, IL-22-PE, IL-17-PerCP FMC008 25 Tests Flow Cytometry Kit Human Myeloid Dendritic Cell 4-Color Contains conjugated antibodies to human BDCA-1/CD1c-APC, CD11c-Fluorescein, BDCA-3/CD141-PE, FMC016 25 Tests Flow Cytometry Kit CD16-PerCP Human Plasmacytoid Dendritic Cell Contains conjugated antibodies to human CD45-APC, BDCA-4/Neuropilin-Fluorescein, BDCA-2/DLEC-PE, FMC017 25 Tests 4-Color Flow Cytometry Kit CD123/IL-3 R-PerCP Mouse Multipotent Mesenchymal Contains conjugated antibodies to mouse Sca-1-APC, CD105-Fluorescein, CD29-PE, CD45-PerCP FMC003 25 Tests Stromal Cell 4-Color Flow Cytometry Kit Mouse Regulatory T Cell 3-Color Contains conjugated antibodies to CD25-PE (Clone 280406), FoxP3-APC (Goat IgG), CD4-PerCP (Clone GK1.5) FMC014 50 Tests Flow Cytometry Kit Rat Regulatory T Cell 3-Color Contains conjugated antibodies to rat FoxP3-APC, CD4-Fluorescein, CD25-PE FMC015 50 Tests Flow Cytometry Kit *Kits also contain appropriate staining buff ers and specifi c isotype controls.

Secondary & Control Antibodies for Flow Cytometry, IHC, & Western Blotting Antibody Controls PRODUCT LABEL TYPE SUGGESTED APPLICATION CATALOG # SIZE Goat IgG Fluorescein Normal goat IgG Control IC108F 200 Tests Goat IgG PE Normal goat IgG Control IC108P 200 Tests Goat IgG PerCP Normal goat IgG Control IC108C 200 Tests Goat IgG None Normal goat IgG Control AB-108-C 1 mg Goat IgG Biotin Normal goat IgG Control BAF108 100 μg

Human IgG1 Fc None NS0-expressed recombinant protein Control 110-HG-100 100 μg Human IgG None Normal human IgG Control 1-001-A 1 mg Sheep IgG None Normal sheep IgG Control 5-001-A 1 mg Sheep IgG APC Normal sheep IgG Control IC016A 200 Tests Sheep IgG Fluorescein Normal sheep IgG Control IC016F 200 Tests Sheep IgG PE Normal sheep IgG Control IC016P 200 Tests

Mouse IgG1 PerCP Normal mouse IgG1, clone # 11711 Control IC002C 200 Tests

Mouse IgG1 APC Normal mouse IgG1, clone # 11711 Control IC002A 200 Tests

Mouse IgG1 None Normal mouse IgG1, clone # 11711 Control MAB002 500 μg

Mouse IgG1 Biotin Normal mouse IgG1, clone # 11711 Control IC002B 200 Tests

Mouse IgG1 Fluorescein Normal mouse IgG1, clone # 11711 Control IC002F 200 Tests

Mouse IgG1 PE Normal mouse IgG1, clone # 11711 Control IC002P 200 Tests

www.RnDSystems.com/go/Secondaries 99 SPECIALIZED TOOLS

Antibody Controls, continued PRODUCT LABEL TYPE SUGGESTED APPLICATION CATALOG # SIZE

Mouse IgG2A None Normal mouse IgG2B, clone # 20102 Control MAB003 500 μg

Mouse IgG2A None Normal mouse IgG2B, clone # 133304 Control MAB0031 500 μg

Mouse IgG2A PerCP Normal mouse IgG2B, clone # 20102 Control IC003C 200 Tests

Mouse IgG2A APC Normal mouse IgG2B, clone # 20102 Control IC003A 200 Tests

Mouse IgG2A Biotin Normal mouse IgG2B, clone # 20102 Control IC003B 200 Tests

Mouse IgG2A Fluorescein Normal mouse IgG2B, clone # 20102 Control IC003F 200 Tests

Mouse IgG2A PE Normal mouse IgG2B, clone # 20102 Control IC003P 200 Tests

Mouse IgG2A Fc None NS0-expressed recombinant protein Control 4460-MG-100 100 μg

Mouse IgG2B None Normal mouse IgG2B, clone # 20116 Control MAB004 500 μg

Mouse IgG2B None Normal mouse IgG2B, clone # 133303 Control MAB0041 500 μg

Mouse IgG2B None Normal mouse IgG2BB, clone # 73009 Control MAB0042 500 μg

Mouse IgG2B APC Normal mouse IgG2B, clone # 133303 Control IC0041A 200 Tests

Mouse IgG2B Biotin Normal mouse IgG2B, clone # 133303 Control IC0041B 200 Tests

Mouse IgG2B Fluorescein Normal mouse IgG2B, clone # 133303 Control IC0041F 200 Tests

Mouse IgG2B PE Normal mouse IgG2B, clone # 133303 Control IC0041P 200 Tests

Mouse IgG2B PerCP Normal mouse IgG2B, clone # 133303 Control IC0041C 200 Tests

Mouse IgG3 None Normal mouse IgG3, clone # 133316 Control MAB007 500 μg

Mouse IgG3 APC Normal Mouse IgG3, clone # 133316 Control IC007A 200 Tests

Mouse IgG3 Fluorescein Normal Mouse IgG3, clone # 133316 Control IC007F 200 Tests

Mouse IgG3 PE Normal Mouse IgG3, clone # 133316 Control IC007P 200 Tests

Mouse IgG3 PerCP Normal mouse IgG, clone # 133316 Control IC007C 200 Tests Rabbit IgG APC Normal rabbit IgG Control IC105A 200 Tests Rabbit IgG Fluorescein Normal rabbit IgG Control IC105F 200 Tests Rabbit IgG PE Normal rabbit IgG Control IC105P 200 Tests Rabbit IgG None Normal rabbit IgG Control AB-105-C 1 mg Rat IgG None Normal rat IgG Control 6-001-A 1 mg

Rat IgG1 None Normal rat IgG1, clone # 43414 Control MAB005 500 μg

Rat IgG1 APC Normal rat IgG1, clone # 43414 Control IC005A 200 Tests

Rat IgG1 Biotin Normal rat IgG1, clone # 43414 Control IC005B 200 Tests

Rat IgG1 Fluorescein Normal rat IgG1, clone # 43414 Control IC005F 200 Tests

Rat IgG1 PE Normal rat IgG1, clone # 43414 Control IC005P 200 Tests

Rat IgG1 PerCP Normal rat IgG1, clone # 43414 Control IC005C 200 Tests

Rat IgG2A None Normal rat IgG2A, clone # 54447 Control MAB006 500 μg

Rat IgG2A APC Normal rat IgG2A, clone # 54447 Control IC006A 50 μg

Rat IgG2A Biotin Normal rat IgG2A, clone # 54447 Control IC006B 50 μg

Rat IgG2A Fluorescein Normal rat IgG2A, clone # 54447 Control IC006F 200 Tests

Rat IgG2A PE Normal rat IgG2A, clone # 54447 Control IC006P 200 Tests

Rat IgG2A PerCP Normal rat IgG, clone # 54447 Control IC006C 200 Tests

Rat IgG2B PerCP Normal rat IgG, clone # 141945 Control IC013C 200 Tests

Rat IgG2B APC Normal rat IgG2B, clone # 141945 Control IC013A 200 Tests

Rat IgG2B Biotin Normal rat IgG2B, clone # 141945 Control IC013B 200 Tests

Rat IgG2B Fluorescein Normal rat IgG2B, clone # 141945 Control IC013F 200 Tests

Rat IgG2B PE Normal rat IgG2B, clone # 141945 Control IC013P 200 Tests

Rat IgG2B None Normal rat IgG2B, clone # 141945 Control MAB0061 500 μg Mouse IgM PerCP Normal mouse IgM Control IC015C 200 Tests Chicken IgY None Normal chicken IgY Control AB-101-C 1 mg

100 For research use only. Not for use in diagnostic procedures. SPECIALIZED TOOLS

NorthernLights™ Secondary Reagents A line of fl uorochrome-conjugated secondary reagents for fl ow cytometry & IHC NorthernLights secondary antibodies are bright and fade-resistant, recognizing mouse, goat, sheep, rat, and rabbit IgG and chicken IgY. Like all R&D Systems antibodies, NorthernLights antibody conjugates are specifi c and deliver a high signal-to-noise ratio. Also available are streptavidin conjugates for labeling biotinylated antibodies.

DESCRIPTION CONJUGATE CATALOG # SIZE Donkey Anti-Goat IgG, Cross-adsorbed NorthernLights-557 NL001 500 μL Donkey Anti-Goat IgG, Cross-adsorbed NorthernLights-637 NL002 500 μL Donkey Anti-Goat IgG, Cross-adsorbed NorthernLights-493 NL003 500 μL Donkey Anti-Rabbit IgG, Cross-adsorbed NorthernLights-557 NL004 500 μL Donkey Anti-Rabbit IgG, Cross-adsorbed NorthernLights-637 NL005 500 μL Donkey Anti-Rabbit IgG, Cross-adsorbed NorthernLights-493 NL006 500 μL Donkey Anti-Mouse IgG, Cross-adsorbed NorthernLights-557 NL007 500 μL Donkey Anti-Mouse IgG, Cross-adsorbed NorthernLights-637 NL008 500 μL Donkey Anti-Mouse IgG, Cross-adsorbed NorthernLights-493 NL009 500 μL Donkey Anti-Sheep IgG, Cross-adsorbed NorthernLights-557 NL010 500 μL Donkey Anti-Sheep IgG, Cross-adsorbed NorthernLights-637 NL011 500 μL Donkey Anti-Sheep IgG, Cross-adsorbed NorthernLights-493 NL012 500 μL Goat Anti-Rat IgG NorthernLights-557 NL013 500 μL Goat Anti-Rat IgG NorthernLights-637 NL014 500 μL Goat Anti-Rat IgG NorthernLights-493 NL015 500 μL Goat Anti-Chicken IgY NorthernLights-557 NL016 500 μL Goat Anti-Chicken IgY NorthernLights-637 NL017 500 μL Goat Anti-Chicken IgY NorthernLights-493 NL018 500 μL Streptavidin NL493 NorthernLights-493 NL997 500 μL Streptavidin NL637 NorthernLights-637 NL998 500 μL Streptavidin NL557 NorthernLights-557 NL999 500 μL NorthernLights Guard Mounting Media NL996 5 mL

Secondary Antibodies ANALYTE LABEL TYPE APPLICATIONS CATALOG # SIZE Chicken IgY None Chicken IgY specifi c polyclonal goat IgG Western blot AF010 500 μg Chicken IgY Biotin Chicken IgY specifi c polyclonal goat IgG Western blot BAF010 250 μg Goat IgG None Goat IgG specifi c polyclonal donkey IgG Western blot AF109 500 μg Goat IgG Biotin Goat IgG specifi c polyclonal donkey IgG Western blot BAF109 250 μg Goat IgG Biotin Goat IgG specifi c polyclonal rabbit IgG Western blot BAF017 250 μg Goat IgG Biotin Goat IgG specifi c polyclonal chicken IgY Western blot BAF019 250 μg Goat IgG HRP Goat IgG specifi c polyclonal donkey IgG Western blot HAF109 1 mL Goat IgG HRP Goat IgG specifi c polyclonal rabbit IgG Western blot HAF017 1 mL Goat IgG HRP Goat IgG specifi c polyclonal chicken IgG Western blot HAF019 1 mL Goat IgG APC Goat IgG specifi c polyclonal donkey IgG Flow Cytometry F0108 100 Tests Goat IgG Fluorescein Goat IgG specifi c polyclonal donkey IgG Flow Cytometry F0109 100 Tests Goat IgG PE Goat IgG specifi c polyclonal donkey IgG Flow Cytometry F0107 100 Tests Goat IgG None Goat IgG specifi c polyclonal rabbit IgG Western blot R-401-C-ABS 500 μg Goat IgG (H+L) PerCP Goat IgG specifi c polyclonal donkey IgG (H+L) chains Flow Cytometry F0124 100 Tests

Hamster IgG APC Monoclonal mouse IgG2B, clone # MAH1.12 Flow Cytometry F0121 100 Tests

Hamster IgG PE Monoclonal mouse IgG2B, clone # MAH1.12 Flow Cytometry F0120 100 Tests

Hamster IgG None Monoclonal mouse IgG2B, clone # MAH1.12 Western blot MAB011 500 μg

Hamster IgG Biotin Monoclonal mouse IgG2B, clone # MAH1.12 Western blot BAM011 250 μg Human IgG Fc None Human IgG Fc specifi c polyclonal goat IgG Western blot G-102-C 100 μg

www.RnDSystems.com/go/Secondaries 101 SPECIALIZED TOOLS

Secondary Antibodies, continued

ANALYTE LABEL TYPE APPLICATIONS CATALOG # SIZE

Hamster IgG Biotin Monoclonal mouse IgG2B, clone # MAH1.12 Western blot BAM011 250 μg Human IgG Fc None Human IgG Fc specifi c polyclonal goat IgG Western blot G-102-C 100 μg

Human IgG1 Fc None Human IgG1 Fc specifi c monoclonal mouse IgG1 Western blot MAB110 500 μg Human IgM None Human IgM specifi c polyclonal goat IgG Western blot G-105-C 100 μg Human IgE None Human IgE specifi c polyclonal goat IgG Western blot G-107-C 100 μg Human IgG (H+L) None Human IgG specifi c polyclonal goat IgG (H+L) chains Western blot G-101-C-ABS 500 μg

Mouse IgG PerCP Mouse IgG specifi c polyclonal goat IgG F(ab')2 (H+L) Fragment Flow Cytometry F0114 100 Tests Mouse IgG None Mouse IgG specifi c polyclonal goat IgG Western blot AF007 500 μg Mouse IgG Biotin Mouse IgG specifi c polyclonal goat IgG Western blot BAF007 250 μg Mouse IgG Biotin Mouse IgG specifi c polyclonal donkey IgG Western blot BAF018 250 μg Mouse IgG HRP Mouse IgG specifi c polyclonal goat IgG Western blot HAF007 1 mL Mouse IgG HRP Mouse IgG specifi c polyclonal donkey IgG Western blot HAF018 1 mL Mouse IgG Fc None Mouse IgG Fc specifi c polyclonal goat IgG Western blot G-202-C 100 μg Mouse IgG None Mouse IgG specifi c polyclonal donkey IgG Western blot D-201-C-ABS2 500 μg

Mouse IgG F(ab’)2 (H+L) APC Mouse IgG specifi c polyclonal goat IgG F(ab’)2 (H+L) Fragment Flow Cytometry F0101B 100 Tests

Mouse IgG F(ab’)2 (H+L) Fluorescein Mouse IgG specifi c polyclonal goat IgG F(ab’)2 (H+L) Fragment Flow Cytometry F0103B 100 Tests

Mouse IgG F(ab’)2 (H+L) PE Mouse IgG specifi c polyclonal goat IgG F(ab’)2 (H+L) Fragment Flow Cytometry F0102B 100 Tests Rabbit IgG None Rabbit IgG specifi c polyclonal goat IgG Western blot AF008 500 μg Rabbit IgG Biotin Rabbit IgG specifi c polyclonal goat IgG Western blot BAF008 250 μg Rabbit IgG HRP Rabbit IgG specifi c polyclonal goat IgG Western blot HAF008 1 mL Rabbit IgG APC Rabbit IgG specifi c polyclonal goat IgG Flow Cytometry F0111 100 Tests Rabbit IgG Fluorescein Rabbit IgG specifi c polyclonal goat IgG Flow Cytometry F0112 100 Tests Rabbit IgG PE Rabbit IgG specifi c polyclonal goat IgG Flow Cytometry F0110 100 Tests Rabbit IgG None Rabbit IgG specifi c polyclonal donkey IgG Western blot D-301-C-ABS2 500 μg Rat IgG HRP Rat specifi c polyclonal goat IgG Western blot HAF005 1 mL Rat IgG None Rat IgG specifi c polyclonal goat IgG Western blot AF005 500 μg

Rat IgG PerCP Rat IgG specifi c polyclonal goat IgG F(ab')2 (H+L) Fragment Flow Cytometry F0115 100 Tests Rat IgG Biotin Rat IgG specifi c polyclonal goat IgG Western blot BAF005 250 μg

Rat IgG F(ab’)2 APC Rat IgG specifi c polyclonal goat IgGF(ab’)2 (H+L) Fragment Flow Cytometry F0113 100 Tests

Rat IgG F(ab’)2 (H+L) Fluorescein Rat IgG specifi c polyclonal goat IgG F(ab’)2 (H+L) Fragment Flow Cytometry F0104B 100 Tests

Rat IgG F(ab’)2 (H+L) PE Rat IgG specifi c polyclonal goat IgG F(ab’)2 (H+L) Fragment Flow Cytometry F0105B 100 Tests Sheep IgG Biotin Sheep IgG specifi c polyclonal donkey IgG Western blot BAF016 250 μg Sheep IgG HRP Sheep IgG specifi c polyclonal donkey IgG Western blot HAF016 1 mL Sheep IgG None Sheep IgG specifi c polyclonal donkey IgG Western blot D-501-C-ABS 500 μg Mouse IgM APC Mouse IgM specifi c polyclonal goat IgG Flow Cytometry F0117 100 Tests Mouse IgM Fluorescein Mouse IgM specifi c polyclonal goat IgG Flow Cytometry F0118 100 Tests Mouse IgM PerCP Mouse IgM specifi c polyclonal goat IgG Flow Cytometry F0119 100 Tests Mouse IgM PE Mouse IgM specifi c polyclonal goat IgG Flow Cytometry F0116 100 Tests Mouse IgM None Mouse IgM specifi c polyclonal goat IgG Western blot G-106-C-ABS 100 μg

102 For research use only. Not for use in diagnostic procedures. SPECIALIZED TOOLS

Staining Reagents for Flow Cytometry PRODUCT DESCRIPTION CATALOG # SIZE FoxP3 Staining Buff er FoxP3 Staining Buff er is formulated and optimized for immunofl uorescent staining of FoxP3+ single-cell FC011 250 mL suspensions intended for fl ow cytometric acquisition and analysis. This product is supplemented with the metabolic inhibitor sodium azide. Flow Cytometry Staining Buff er (1X) For immunofl uorescent staining of single-cell suspensions and subsequent dilution, suspension, FC001 500 mL washing, and storage of cells intended for fl ow cytometric acquisition and analysis. Flow Cytometry Human Lyse Buff er (10X) Formulated and optimized to prepare human whole blood samples or any other cell preparation con- FC002 50 mL taining an excess of human red blood cells, intended for fl ow cytometric acquisition and analysis. Flow Cytometry Mouse Lyse Buff er (10X) Formulated and optimized to prepare mouse samples that contain an excess of red blood cells, such as FC003 50 mL spleen or whole blood samples, intended for fl ow cytometric acquisition and analysis. Flow Cytometry Fixation Buff er (1X) For fi xation and storage of single-cell suspensions intended for subsequent intracellular fl ow FC004 100 mL cytometry applications. Flow Cytometry Permeabilization/Wash Buff er For permeabilization of single-cell suspensions, dilution, and washing of these samples for subsequent FC005 250 mL I (1X) intracellular fl ow cytometry applications. Flow Cytometry Fixation & Permeabilization Formulated and optimized for simultaneous fi xation and permeabilization of single-cell suspensions, FC007 125 mL Buff er I (1X) dilution, and washing of these samples for subsequent intracellular fl ow cytometry applications. Flow Cytometry Fixation & Permeabilization All buff ers required for the fi xation and permeabilization of single-cell suspensions, dilution, and FC009 1 Kit Buff er Kit I washing of these samples for subsequent intracellular fl ow cytometry applications (100 mL of Fixation Buff er and 250 mL of Permeabilization/Wash Buff er). Avidin-Fluorescein For use as a secondary reagent in fl ow cytometry in conjunction with biotinylated primary labeling F0030 100 Tests reagents. Streptavidin-Allophycocyanin For use as a secondary reagent in fl ow cytometry in conjunction with biotinylated primary labeling F0050 100 Tests reagents. Streptavidin-Phycoerythrin For use as a secondary reagent in fl ow cytometry in conjunction with biotinylated primary labeling F0040 100 Tests reagents.

Molecular Weight Markers MARKER DESCRIPTION CATALOG # SIZE Biotinylated MW Markers A mixture of seven individually biotinylated proteins, rat Phosphorylase-b (122 kDa), bovine Catalase MW001 500 μL (65 kDa), recombinant human Annexin V (36 kDa), recombinant human IL-6 (21 kDa), recombinant human TNF- (17.5 kDa), bovine Cytochrome c (10 kDa), and recombinant human EGF (6.2 kDa), and one blue dye-conjugated protein, recombinant human Annexin V (43 kDa) for use in Western blot. Prestained MW Markers A mixture of seven individually blue dye-conjugated proteins, rat Phosphorylase-b (122 kDa), BSA (94 MW002 500 μL kDa), bovine Catalase (65 kDa), recombinant human Annexin V (39 kDa), Trypsin Inhibitor (23 kDa), recombinant human TNF- (19 kDa) and Aprotinin (7 kDa) for use in Western blot.

www.RnDSystems.com/go/Secondaries 103 SPECIALIZED TOOLS

Staining Kits & Reagents for Cells & Tissues Cell and Tissue Staining Kits are intended for localization of antigens in a broad range of histological and cytological specimens. These kits are based on the formation of the Avidin-Biotin Complex (ABC) with primary antibodies that react with the tissue antigens under study. The kits are made in ready- to-use format including prediluted Secondary Biotinylated Antibodies, and High Sensitivity Streptavidin-conjugated Horseradish Peroxidase (HSS- HRP) that eliminates the need for extra steps in the procedure, minimizes hands-on time, maximizes convenience, and reduces the risk of erroneous calculations.

Cell & Tissue Staining Kits SPECIES LABEL COMPONENTS CATALOG # SIZE Anti-goat HRP-DAB System Secondary Biotinylated Antibody, Streptavidin-HRP Conjugate, DAB Chromogen, CTS008 50 Tests Anti-mouse DAB Chromogen Buff er, Blocking Reagents CTS002 Anti-rabbit CTS005 Anti-rat CTS017 Anti-sheep CTS019 Anti-goat HRP-AEC System Secondary Biotinylated Antibody, Streptavidin-HRP Conjugate, AEC Chromogen, CTS009 50 Tests Anti-mouse AEC Chromogen Buff er, Blocking Reagents CTS003 Anti-rabbit CTS006 Anti-rat CTS018 Anti-sheep CTS020

Cell and Tissue Staining Reagents PRODUCT DESCRIPTION CATALOG # SIZE Basic Antigen Retrieval Reagent Antigen retrieval system that utilizes a heat-induced recovery of cell and tissue antigens CTS013 50 mL

Acidic Antigen Retrieval Reagent Antigen retrieval system that utilizes a heat-induced recovery of cell and tissue antigens CTS014 50 mL

Universal Antigen Retrieval Reagent Antigen retrieval system that utilizes a heat-induced recovery of cell and tissue antigens CTS015 50 mL

Antigen Retrieval Reagent Sampler All three reagents (CTS013, CTS014, CTS015) in one convenient pack CTS016 50 mL of each

Avidin-Fluorescein For use as a secondary reagent in immunofl uorescent assays in conjunction with biotinylated primary F0030 100 Tests labeling reagents Streptavidin-Allophycocyanin For use as a secondary reagent in immunofl uorescent assays in conjunction with biotinylated primary F0050 100 Tests labeling reagents Streptavidin-Phycoerythrin For use as a secondary reagent in immunofl uorescent assays in conjunction with biotinylated primary F0040 100 Tests labeling reagents Streptavidin-Alkaline Phosphatase For use in ELISA or Western blot assays AR001 1 mL

DAB Enhancer DAB Enhancer is a concentrated 50X metal based solution used to intensify the DABChromogen CTS010 3 mL (Catalog Numbers # CTS002, CTS005, CTS008, CTS017, CTS019). In the presence of the DAB Enhancer, the DAB Chromogen produces a clear brown-black staining with minimal background Mounting Medium Permanent aqueous mounting medium CTS011 15 mL

NorthernLights™ Guard Mounting Media Ready to use mounting medium that signifi cantly extends the light emitting capacity of fl uorescent dyes NL996 5 mL

104 For research use only. Not for use in diagnostic procedures. SPECIALIZED TOOLS

CHROMATIN IP KITS & MODULES Chromatin IP Kits R&D Systems ExactaChIP™ Chromatin Immuoprecipitation (ChIP) Kits are designed to provide a fast, simple method for the identifi cation of genomic DNA target sequences bound by a specifi c protein. Protein-DNA complexes are fi xed by formaldehyde crosslinking, the chromatin is sheared, and the complex is immunoprecipitated using an antibody specifi c for the target protein. Protein-bound DNA fragments are purifi ed and detected by standard PCR. ExactaChIP Kits may be used to identify conditions or treatments that activate the transcription factor of interest and to analyze regions in the DNA where the transcription factor binds. The antibodies provided in the kits have been validated for ChIP yielding rapid and reliable results. Kit Components • Analyte-specifi c primary antibody • Biotinylated control antibody • Chelating resin solution • Control primer set • Lysis buff er • Wash buff ers • Dilution buff er • Protocol and troubleshooting guide

CHIP KIT ANTIBODY SPECIES CONTROL PRIMERS CATALOG # CHIP KIT ANTIBODY SPECIES CONTROL PRIMERS CATALOG # Acetyl-Histone H3 Human N/A ECP5214 c-Myc Human p21 ECP3696 (K9/K14) Nanog Human Nanog ECP1997 Acetyl-Histone H4 Human N/A ECP5215 (K4/K7/K11/K15) NFB1 Human p21 ECP2697 BMI-1 Human HOXC13 ECP3334 NFB2 Human p21 ECP28881 Brachyury Human VEGF ECP2085 Notch-1 Human c-Myc ECP3647

-Catenin Human SU(Z)12 ECP1329 Oct-3/4 Human/Mouse Nanog ECP1759 c-Rel Human p21 ECP2699 Olig2 Human p21 ECP2418 CREB Human fos ECP2989 p53 Human/Mouse p21 ECP1355 FoxP3 Human IL-2 ECP3240 p300 Human fros ECP3789

GATA-4 Human mucin4 ECP2606 RelA Human p21 ECP5078 GATA-5 Human mucin4 ECP2170 RUNX2/CBFA1 Human VEGF ECP2006 GATA-6 Human mucin4 ECP1700 Smad2/3 Human E-cadherin ECP3797 GLI-1 Human Bcl-2 ECP3324 Smad4 Human p21 ECP2097 GLI-2 Human Bcl-2 ECP3526 Snail Human E-cadherin ECP3639 GLI-3 Human gli-1 ECP3690 SOX2 Human/Mouse Nanog ECP2018 SOX17 Mouse Lama1 ECP1924 HIF-1 Human/Mouse Erythropoietin, VEGF ECP1935 STAT3 Human/Mouse c-Myc ECP1799 HIF-2 Human Oct-3/4 ECP2886 Ikaros Human VPAC ECP4984 STAT5a/b Human/Mouse Bcl-x ECP2168 KLF4 Human B2R ECP3640 T-bet/TBX21 Human IFN- ECP5385 KLF4 Mouse B2R ECP3158

Chromatin IP Modules R&D Systems ExactaChIP Chromatin Immunoprecipitation (ChIP) Modules contain a biotinylated antibody in suffi cient quantity to process 20 samples for ChIP. Also included in the module are a negative control antibody and primer pairs complementary to DNA regions known to associate with the transcription factor or nuclear protein of interest.

MODULE SPECIES CATALOG # SIZE MODULE SPECIES CATALOG # SIZE

-Catenin Human MECP1329 1 Kit Oct-3/4 Human/Mouse MECP1759 1 Kit GLI-1 Human MECP3324 1 Kit p53 Human/Mouse MECP1355 1 Kit

HIF-1 Human/Mouse MECP1935 1 Kit Smad4 Human MECP2097 1 Kit HMGB1 Human MECP1690 1 Kit SOX2 Human/Mouse MECP2018 1 Kit KLF4 Human MECP3640 1 Kit STAT3 Human/Mouse MECP1799 1 Kit Nanog Human MECP1997 1 Kit

www.RnDSystems.com/go/ChIP 105 NOTES

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106 For research use only. Not for use in diagnostic procedures. NOTES

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www.RnDSystems.com 107 NOTES

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108 For research use only. Not for use in diagnostic procedures. ORDERING INFORMATION

CUSTOMERS IN USA & CANADA Online ordering is available for customers in the USA and Canada. After fi nding your products, click the shopping cart symbol to create an account or to make an online purchase.

Website: www.RnDSystems.com Phone: 1-800-343-7475 Fax: 1-612-656-4400 Mail: R&D Systems, Inc. 614 McKinley Place NE Minneapolis, MN 55413

INTERNATIONAL DISTRIBUTORS

Please visit www.RnDSystems.com/go/Distributors for a full list of international distributors.

FOR ALL PURCHASES, PLEASE INCLUDE THE FOLLOWING INFORMATION WITH YOUR ORDER

• Name & Contact Information • Name of Institution • Delivery Address • Invoice Address • Purchase Order Number* or Credit Card Number • Quantity and Product Size • Product Catalog Number and Description • Name of Principal Investigator (optional) • Telephone Number • E-mail Address (optional)

For European Customers: VAT Exemption Certifi cate (UK) or E.C. equivalent

* New customers wishing to use a P.O. number, please call customer service at 1-800-343-7475 to set up an account. R&D Systems Products for Neuroscience Research The R&D Systems Family off ers a wide range of high quality products for neuroscience research.

High performance antibodies, and the most referenced collection of bioactive proteins & immunoassays in the industry. www.RnDSystems.com

A unique selection of chemically-based small compounds and peptides, including neurotoxins and caged compounds, to agonize or inhibit neuronal proteins & pathways. www.tocris.com

The leading source of enzymes, inhibitors, and substrates for the study of autophagy & the ubiquitin proteasome pathway. www.bostonbiochem.com

USA & Canada R&D Systems, Inc. Europe R&D Systems Europe Ltd. China R&D Systems China Co., Ltd. 614 McKinley Place NE 19 Barton Lane, Abingdon Science Park 24A1 Hua Min Empire Plaza Minneapolis, MN 55413, USA Abingdon OX14 3NB, UK 726 West Yan An Road, Shanghai, PRC 200050 TEL: (800) 343-7475 (612) 379-2956 TEL: +44 (0)1235 529449 TEL: +86 (21) 52380373 FAX: (612) 656-4400 FAX: +44 (0)1235 533420 FAX: +86 (21) 52371001 E-MAIL: [email protected] E-MAIL: [email protected] E-MAIL: [email protected] www.RnDSystems.com www. RnDSystems.com www.RnDSystemsChina.com.cn

International Distributors Please visit www.RnDSystems.com/go/Distributors for a full list of international distributors.

CA112_neurosci_OCT