Notch Signaling Pathway Important for Cancer & T Cell Research

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The Importance of Notch CONTENTS

The highly conserved Notch signaling pathway regulates many different cell fate deci- Notch Scientific Relevance 1–3 sions in both vertebrate and invertebrate species. It is important for pattern formation Notch Signaling, Notch & Diseases, during development such as neurogenesis, angiogenesis or myogenesis and regulates Notch & Cancer, Notch & Innate and T cell development and stem cell maintenance [1]. Notch signaling is also involved in Adaptive Immunity cellular processes throughout adulthood [2]. Signaling via Notch occurs between receptors and its ligands, both at the surface of neighbouring cells (see Figure 1, Notch Re- Notch Receptors ceptors and Their Ligands). In mammals, expression of four Notch receptors (Notch1–4) Notch1 & Notch2 4 and five canonical ligands [Delta-like ligand (DLL1, 3, 4) and Jagged (Jagged-1, -2) coordinate activation of this signaling pathway [3]. Antibodies & Proteins

Canonical Notch Ligands 4–5 DLL1, DLL3, DLL4, Jagged-1 and -2 TMD Antibodies & Proteins RAM NRR MNNL TMD LNR EGF-like Repeats SP SP DSL EGF CR PDZ ANK HD Notch1 P TAD Jagged-1 & -2 Non-Classical Notch Ligands 6 NLS DOS DLL1 DLK1 and DLK2 Antibodies, Notch2 TAD Proteins and ELISA Kit DLL3

Notch3 DLL4 Non-Confirmed Notch Ligand 6

DLK1 & 2 DNER Antibodies & Proteins Notch4 Plasma Membrane Plasma Membrane Notch Target HES1 7

E3UBLs and DUBs 7 FIGURE 1: Notch Receptors and their Ligands. Mammals possess four Notch receptors (Notch1–4) and five ligands including Jagged-1 and -2 and Delta-like (DLL) 1, 3 and 4. Additional noncanonical Notch ligands are DLK1, DLK2. ADAM17 Blocking Antibody 8 ANK: Ankyrin Repeats; CR: Cysteine-rich Domain: DOS: Delta and OSM-11-like Proteins Domain; DSL: Delta, Serrate and LAG-2 Domain; EGF: Epidermal Growth Factor-like Repeats; HD: Heterodimerization Domain; LNR: Cysteine-rich Lin12-Notch Repeats; NRR: Negative Regulatory Region; MNNL: Module at N-terminal Domain of Notch Ligands; NLS: Nuclear Localization Signal; P: PEST Domain; PDZ: PDZ Domain; PM: Notch Processing / Plasma Membrane; RAM: RBPJ-associated Molecule; SP: Signal Peptide; TAD: Transactivation Domain; TMD: Transmembrane Domain J-Secretase Inhibitors 8

Adapted from: The intracellular region of Notch ligands: does the tail make the difference? A. Pintar, et al.; Biol. Direct 2, 19 (2007), The canonical Notch signaling pathway: unfolding the activation mechanism: R. Kopan & M. X. Ilagan; Cell 137, 216 (2009)

Full Panel of Products! Full Panel of Products Inside! See Antibodies –Antibodies Recombinant – Recombinant Proteins – Proteins ELISA Kits– ELISA – Small Kits – Molecules Small Molecules Inside Notch Receptors and Ligands Family

Mammalian Notch receptor homologs (Notch1 to 4) encode a Notch extracellular domain (NECD) that binds ligands, a transmembrane domain, and a Notch intracellular domain (NICD) that translocates to the nucleus to serve as a transcriptional cofactor. Mam- malian NECDs consist of 29 to 36 EGF repeats followed by three Lin–Notch repeats (LNRs). EGF11 and 12 domains alone are suffi- cient for binding to Notch ligands (Jagged/DLL). All canonical Notch ligands are transmembrane proteins that share a largely simi- lar structure, with an extracellular domain comprised primarily of multiple EGF repeats (6 for DLL3; 8 for DLL1 and DLL4; or 16 for Jagged-1 and Jagged-2), followed by “module at the N-terminus of Notch ligands” (MNNL) domain and by a “Delta/Serrate/Lag-2 (DSL) domain [1]. The non-canonical Notch ligands lack the DSL domain, among these are proteins delta homolog 1 and 2 (DLK1and DLK2) [4]. Some proteins including Contactin-3 and -6 and DNER have been postulated to act as Notch ligands, but confirmation of these observa- tions are still needed [5].

Activation of Notch Signaling Recycling Transcriptional Clathrin/Epsin- Lysosomal in Vesicles Effects, Cell Adhesion, Degradation Migration & Oncogenic dependent Transformation Trans-Endocytosis The Notch receptors are synthesized as single precur- Clathrin 5DE 5DE Ub sor proteins that are cleaved during transport to the cell 0LQG%RPE (SVLQ 5DE CME PDZ Proteins (8ELTXLWLQ/LJDVH surface (at cleavage site S1, not shown in the Figure 2), Clathrin Ubiquitination Ub (SVLQ (SVLQ where they are expressed as heterodimers. Notch signal transduction is initiated upon binding of a Notch receptor Notch Ligand Pulling Force Notch QHHGHGIRU6FOHDYDJH Ligand heterodimer to a ligand located on a neighbour cell (see Blocking Ligand Antibodies Receptor Figure 2: Notch Signaling Pathway). Upon receptor-ligand Inhibitory NECD NNR Domain Recombinant (Notch binding, ubiquitination by RING E3 ligases (such as Mind Receptors & Extracellular Trans-Interaction Ligands Domain) bomb (Mib) or Neuralized), marks the ligands for Epsin-

ADAM10/17 Blocking Antibodies dependent endocytosis. This event generates a mechani- (S2 Cleavage) D-Secretase Inhibitors cal pulling force, which drives conformational changes of

Ligand Extracellular Department Interaction the Notch receptor and facilitates its sequential proteo- Proteolysis

Ubiquitination Transmembrane lytic cleavages [3]. The cleavage (at S2 site) which is trig- Ub Notch gered by ligand binding and mediated by a disintegrin and 1RWFK5HFHSWRU P 1RWFK5HFHSWRU +HWHURGLPHU Clathrin metalloproteinase (ADAM family, also called TACE, tumor Phosphorylation $3 +HWHURGLPHU aPKC] Dynamin necrosis factor-D-converting enzyme) family peptidase, 1HGG Clathrin- Nicastrin (8ELTXLWLQ Numb dependent J-Secretase Inhibitors Presenilin /LJDVH releases the NECD, whereas the cleavage (at S3 /S4 sites) J-Secretase Modulators $3+ Endocytosis 3(1 J-Secretase- mediated by J-secretase activity of a multiprotein com- Complex Early (S3/S4 Cleavage) Endosome Acidification plex (consisting of presenilin, nicastrin, APH1 and PEN2) Inhibitors Polyubiquitination releases the NICD. The Notch intracellular domain trans- Proteasomal Degradation Ub 'HOWH[,7&+>$,3@1HGG Ub Ub Numb Lysosomal locates to the nucleus where it binds with CSL/Rbpj (re- Ub (8ELTXLWLQ/LJDVHV Degradation Ub combination signal binding protein for immunoglobulin N Deubiquitination

NICD H,)) Signal-receiving Cellj region) and recruits a transcriptional Ligand-expressing Cell complex to activate P (Notch Phosphorylation *6.E siRNA, microRNA the transcription of downstream targets, including Hairy/ Proteasome Intracellular CDK8 to target mRNAs Inhibitors Domain) enhancer-of- split (Hes) and Hes-related with YRPW motif protein (Hey) family genes [6]. Activity of Notch receptors and ligands is profoundly affected by glycosylation of EGF NICD Transcription Complex Inhibitors repeats in the extracellular domain. O-fucosyltransferases, which add fucose to serine and threonine residues and O- Notch Target &R$ 0$0/ Genes Active glucosyltransferases, which add glucose to serine residues, &R5 CSL Notch Target Genes NICD +(6 followed by extension of the sugar by Fringe family Glc- 5HSUHVVHG + NICD +(< 0\F NAc-transferases are essential for modulating the binding CSL %FO &\FOLQ' avidity of ligand-receptor pairs. Other post-translational Nucleus ,5) ... events, including mono- or polyubiquitination by specific E3 ubiquitin ligases and phosphorylation as well as endo- FIGURE 2: Simplified Notch Signaling Pathway, including potential therapeutic target possibilities. cytic trafficking, regulate the activities of both the Notch NUCLEUS: Co-A: Co-Activator Proteins; Co-R: Co-Repressor Proteins; CSL: CBF1/Su(H)/LAG-1 Complex; MAML1: receptors and their ligands. Mastermind-like 1. SIGNALING: AP2: Adaptor Protein 2; E3 Ubiquitin Ligases: Deltex, ITCH, Nedd4; eIF3F: Eukaryotic Translation Initiation Factor 3 Subunit F; NRR: Negative Regulatory Region

Notch and Diseases

The Notch pathway plays an important role in many different processes in a wide range of tissues and deregulations in Notch signaling components have been associated with various human disorders such as cancer, immune disorders, developmental syndromes, stroke and cognitive symptoms. Other disorders affecting vertebral column such as scoliosis or the vasculature, hypertension and the developmental disorder Alagille syndrome are also caused by Notch defects [7].

2 www.adipogen.com Notch and Cancer

Components of the Notch signaling pathway are altered in diseases and cancers (T and B cell lymphoproliferative disorders, liver, breast, brain, bladder, lung and prostate). Notch can act either as an oncogene or tumor suppressor depending on the cellular con- text. Components of the Notch signaling are not frequently mutated in most tumor types, although mutations appear to accumu- late during growth of tumors. However, there are exceptions with loss-of-function mutations in Notch receptors supporting their tumor-suppressive role in multiple malignancies, including bladder cancer and squamous cell carcinoma. Constitutive activation of the Notch receptors through gene rearrangements or gain-of-function mutations leads to Notch receptors' oncogenic function in T cell acute lymphoblastic leukemia, in chronic lymphocytic leukemia and in solid tumors such as lung adenocarcinoma. In breast and prostate cancer, Notch signaling frequently appears to be upregulated, and high levels of Jagged-1 expression correlate with poor prognosis of some tumors showing that the level of Notch signaling is critical in regulation of cell proliferation, survival or death. Given that Notch signaling is dysregulated in different types of cancer, Notch inhibitors alone or in combination with chemothera- peutics are currently clinically evaluated and become an exciting new approach to fight cancer (see Figure 2).

Notch and Regulation of Innate and Adaptive Immunity

Notch signaling plays an essential role during development and differentiation of hematopoietic cells [8]. During early stages of T cell development, Notch Group 3 ILC is required continuously in the thymus while in the bone marrow, it inhibits B cell development. Notch also plays essential roles later during lymphocyte de- ILC1 (e.g. NK cells) velopment, in particular during T cell lineage com- mitment and maturation in the thymus and dur- Group 3 ILC ing marginal zone B (MZB) cell development in the HSC spleen. Notch is also a key factor in dendritic cell Notch ILC2 (e.g. Nuocytes) (DC) homeostasis. Finally, Notch functions in the de- + velopment of the newly described Innate Lymphoid RORD Cells (ILCs) playing roles in innate immune responses Group 3 ILC to infectious microorganisms, in the genera tion of RORD+ IL-17 producing ILC secondary lymphoid organs and in tissue remod- CLP eling after tissue injury or infection (see Figure 3).

RORJl– AI IR NKp46+ Notch Notch Notch FIGURE 3: The role of Notch Signaling in the development of innate lymphoid IL-22 producing ILC cells. Haematopoietic stem cell (HSC)-derived common lymphoid progenitors (CLPs) give rise to adaptive immune cells, such as T cells and B cells, as well as to innate lymphoid + cells (ILCs). ILCs function in innate immune responses and are grouped into three RORJ LTi Cell major classes: group 1, group 2 and group 3. ILCs diverge in their requirement for Notch (as indicated). AHR: aryl hydrocarbon receptor; IL: interleukin; LTi: lymphoid + tissue-inducer; NK: natural killer; ROR: retinoid-related orphan receptor. B Cell T Cell D1E7 RORJl– Adapted from: Regulation of innate and adaptive immunity by Notch: F. Radtke, et al.; Nat. Rev. Immunol. 13, 427 (2013)

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NotchNotch Signaling Signaling P y Pathway www.adipogen.com Activation, Signaling & Regulation TMD NRR

RAM SP ll eats LNR EGF-like Rep ANK HD Notch1 P TAD

Ub NLS eptors Clathrin/Epsin-mediated Binding to Degradation Notch2 TAD

Endocytosis Rec Recycling Transcriptional PDZ-containing in Endosome Proteins Dynamin Effects, Cell Adhesion, Neur Lysosome (E3 Ubiquitin Ligase) Notch3 Migration & Oncogenic PDZ Notch Clathrin Endosome Proteins Rab5 Transformation Ub Rab4 Epsin Notch4 REFERENCES Rab11 CME Ligand/NECD Mind Bomb 1 Trans-Endocytosis Clathrin Plasma Membrane Notch (E3 Ubiquitin Ligase) Ligands Ub Epsin TMD Ubiquitination Epsin MNNL Notch PDZ DSL EGF CR Ligand Cis-Interaction SP Jagged-1 & -2 (Inhibitory: Binding DLL1, 4 DOS

Receptor & Ligand gands Jagged-1, -2 DLL1 NECD on same Cell) Exosomes Trans-Interaction DLL3 (Activation) Pulling Force (Notch NotchR 1-4 (needed for S2 Cleavage) Unliganded DLL1, 3, 4 Extracellular DLL4 Li Notch [1] Domain) Receptor Jagged-1, -2 Pulling Force DLK1, 2 Notch signaling at a glance: K. Hori, et al.; J. Cell Sci. 126, 2135 NotchR 1-4 from Ligand DLK1 & 2 ADAM10/17

(S2 Cleavage) Space Extracellular Plasma Membrane LJQDO Clathrin 1/6 1XFOHDU /RFDOL]DWLRQ6

Ligand omain ins Ub AAK1 Ub AP2 ANK: Ank yrin Repeats P: PEST D Interaction Proteolysis Ubiquitination Ub n Ub P Clathrin CR: Cyst eine-rich Domai PDZ: PDZ Domain P Domain cule P lta and OSM-11-like Proteins sociated Mole [2] P AP2 DOS: De RAM: RBPJ-as -2 Domain l Peptide DSL: Delta, Serrate and LAG ts SP: Signa Hematopoietic stem cells: to be or Notch to be: A. Bigas Transmembrane Signaling rmal Growth Factor-like Repea sactivation Domain Degradation TAD: Tran t Nedd4 Dynamin LNX EGF: Epide DLQ Q'RP Notch Receptor Notch Receptor Inactivation +' +HWHURGLPHUL]DWLR TMD: Tra nsmembrane Domain Cytoplasm (Heterodimer) (E3 Ubiquitin (E3 Ubiquitin ch Repeats Numb ine-rich Lin12-Not Doma Notch Ligase) Ligase) Dynamin LNR: Cyste Proteasomal Notch NRR: Negative Regulatory Region Notch Receptor Phosphorylation Rab5/11 l Domain of Notch Ligands r) Degradation D3.&] Receptor ule at N-termina (Heterodime Clathrin- Syntaxin-7 MNNL: Mod J-Secretase Complex Recycling (S3/S4 Cleavage) mediated Proteases Polyubiquitination (via Trans Binding [3] Ub Endocytosis Glycosylation Numb Golgi Network) nical & L. Espinosa; Blood 119, The Notch signalling sys- Ub Deltex, ITCH [AIP4], Nedd4 [CME] Modification Notch Cano Ub (E3 Ubiquitin Ligases) Ub Ubiquitination Pathway

t Processes Fringe-mediated Ub Lysosome ion Translocation Glycosylation Trans Ub Early P Phosphorylat Golgi Cell Signal-receiving Ce Ligand-expressing NICD Deubiquitination P Endosome Network ontaining Protein (Notch Metalloproteinase Domain-c P eIF3F Ub ADAM: A Desintegrin and Furin-like Protease Degradation 2 Intracellular Deltex Early Endosome HOPS/ AP2: A daptor Protein Phosphorylatio Protein Kinase 1 (S1 Cleavage) (E3 Ubiquitin Syntaxin-17 AKK1: AP 2-associated F Domain) n Ligase) Mono- ion Initiation Factor 3 Subunit GSK3E ITCH eIF3F: Euk aryotic Translat ansport tem: recent insights into the complexity of a conserved pathway: K.G. Ubiquitination Cleavage ting Complexes Required for Tr CDK8 ESCRT ESCRT: Endosomal Sor Protein Sorting Complex ? motypic Fusion and Vacuole

HOPS: Ho ossary grin-linked Kinase

Bcl-2 Rab7 ILK: Inte Gl Canonical E3 Ubiquitin and of Numb Protein X Nucleus Non-Canonical NB LNX: Lig Proteins NF- Ligases ne Double Minute 2 Co-A: Co-Activator Signaling Signaling MDM2: Muri essor Proteins PI3K/Akt/TOR es Co-R: Co-Repr MVBs: M ultivesicular Bodi Lag-1 Complex O-Fucosylation -Catenin Ub G CSL [RBP J]: CBF1/Su(H)/ E 1HXU 1H XUDOL]H like 1 Endoplasmic Ub Late Endosome/ MAML1: Mastermind- [4] of NECD Rumi S1: P rotease Site 1 Reticulum Polyubiquitination Ub MVBs Guruharsha, et al.; Nat. Rev. Genet. 9, Possible roles of (Notch Extracellular POFUT-1 of Notch Receptor t Domain) MDM2 Ligand-independent Activation Cancers FOHXNHPLD 7$// ĺ7FHOODFXWHO\PSKREODVWL Rheostat VRUGHUV WFKOHYHOVUHJXODWHVLJQDOLQJRXWSXW ĺ%FHOOO\PSKRSUROLIHUDWLYHGL Proteasomal ĺ'LIIHUHQWLQWHQVLWLHVRI1R DOPDOLJQDQFLHV e ULHW\RIVWHPFHOOW\SHV ĺ2WKHUKHPDWRORJLF OOREODVWRPDV Physiological Relevanc ĺ,QIOXHQFHVFHOOIDWHRIDYD RJHQHVLV VLQFOXGLQJJOLRPDVDQGPHGX Degradation HSDWWHUQLQJDQGPRUSK ĺ%UDLQWXPRU SURVWDWHDQGRWKHURUJDQV ĺ&RQWULEXWHVWRWLVVX WEODGGHUOXQJ nsition [EMT] tion, proliferation, survival ĺ6ROLGWXPRUVLQOLYHUEUHDV DLK1 in the Notch pathway during development and disease: F.A. Epithelial-Mesenchymal Tra ĺ by affecting cell differentia RI(07 ĺ0HODQRPDV ĺ3URPRWLRQ ĺ and apoptosis PRUV ĺ&RORUHFWDOWX UVWHPFHOOV ĺ(PEU\RQDOGHYHORSPHQW ODQGUHQHZDORIFDQFH Hematopoiesis 6& ĺ5HJXODWHVVXUYLYD ĺ&DUGLDFGHYHORSPHQW LFVWHPFHOOV + ĺ*HQHUDWLRQRIKHPDWRSRLHW HQW Co-A ĺ3URPRWLRQRIPHWDVWDVLV HYHUDOVWDJHVRI7FHOOGHYHORSP NICD HES-1 ĺ&RQWUROVV Angiogenesis/Heart RIEORRGYHVVHOV Notch Target WRI%FHOOVLQPDUJLQDO]RQH KHVSURXWLQJSDWWHUQ PHQW HEY-1 m ĺ'HYHORSPHQ &V ĺ&RQWURORIW DQGYDVFXODUGHYHORS 7 FBXW7/ Genes Central Nervous Syste WRILQQDWHO\PSKRLGFHOOV ,/ XODWRURIWXPRUDQJLRJHQHVLV P FLW\ ĺ'HYHORSPHQ ĺ3LYRWDOUHJ SEL10 Myc PHQW SODVWL 201 ĺ&16GHYHORS ĺ&UXFLDOLQKHDUWGHYHORPHQW VHDVHV [5] (E3 Ubiquitin Bcl-2 QLWRUFHOOV 13& FKDVELFXVSLGDRUWLFYDOYHGL CSL MAML1 Co-R ĺ1HXUDOSURJH Metabolism ĺ&RQJHQLWDOKHDUWGHIHFWVVX

Ub Ligase) newal IZKLWHDQGEHLJHDGLSRF\WHV ULRSDWK\ZLWKVXEFRUWLFDO MAY 1822, Cyclin D1 maintenance and self-re ĺ5HJXODWHVWKHSODVWLFLW\R XWRVRPDOGRPLQDQWDUWH Falix, et al.; Biochim. Biophys. Acta Delta/Notch- ĺ&HUHEUDOD Ub RGXFWLRQ t ĺ1HXULWHRXWJURZWK JOXFRVHSU eukoencephalopathy ĺ$IIHFWVKHSDWLF Ub IRF6 HQLWRUVLQWRDVWURF\WHV OHKRPHRVWDVLV ĺ infarct and l + NICD ĺ'LIIHUHQWLDWLRQRIJOLDSURJ ĺ,QYROYHGLQVNHOHWDOPXVF Ub Poly- GLVHDVH lls) Co-A ĺ,QYROYHPHQWLQ$O]KHLPHU¶V ĺ (by regulating satellite ce Co-R SHFLILFDWLRQ ubiquiti- Notch Target ĺ5HJXODWHV0YHUVXV0PDFURSKDJHVV nation CSL Genes Repressed

www.adipogen.com Nucleus Like EGF-Related Receptor (DNER) Is Not a Notch Ligand: M. Greene, Phosphorylation ILK et al.; PLoS One 11,F t[6] Notch signalling in the nucleus: roles of Mastermind-like (MAML) transcriptional coactivators: M. Kitagawa; J. Biochem. 159, t[7] Therapeutic modulation of Ask for our detailed Notch Signaling Wallchart Notch signalling-are we there yet? E.R. Andersson & U. Lendahl; Nat. Rev. Drug Discov. 13, t[8] Regulation of innate and adaptive or download it from www.adipogen.com immunity by Notch: F. Radtke, et al.; Nat. Rev. Immunol. 13, 427 (2013)

3 For updated prices and additional information visit www.adipogen.com or contact your local distributor. Notch Receptors Notch1 & Notch2

ANTIBODIES PID SIZE ISOTYPE APPLICATION SPECIES anti-Notch1 (mouse), mAb (22E5) AG-20B-0051 100 μg Rat IgG2aN FACS Ms anti-Notch1 (mouse), mAb (22E5) (Biotin) AG-20B-0051B 100 μg Rat IgG2aN FACS Ms anti-Notch2, mAb (16F11) AG-20B-0052 100 μg Rat IgG1N FACS Ms anti-Notch2, mAb (16F11) (Biotin) AG-20B-0052B 100 μg Rat IgG1N FACS Ms

PROTEINS PID SIZE SOURCE ENDOTOXIN SPECIES Notch1 (mouse):Fc (human) (rec.) AG-40B-0109 50 μg | 3 x 50 μg CHO cells <0.1EU/μg Ms Notch2 (mouse):Fc (human) (rec.) AG-40B-0110 50 μg | 3 x 50 μg CHO cells <0.01EU/μg Ms

Notch1 Notch2 FIGURE: Detection of endogenous mouse Notch1 or Notch2 on resting and activated T cells with anti-Notch1 (mouse), mAb (22E5) (Prod. No. AG-20B-0051) and anti-Notch2, mAb (16F11) (Prod. No. AG-20B-0052), respectively.

METHOD: CD4+ T cells from C57BL/6 mice were treated with anti-CD3 on plastic (solid line), IL-2 (dotted line) or medium alone as a negative control (shaded histogram) for 24h. The staining was revealed with a secondary anti-mouse IgG-PE (1/200) and then analyzed by flow cytometry.

Canonical Notch Ligands

Notch Ligand DLL1 (Delta-like Protein 1) BULK

ANTIBODIES PID SIZE ISOTYPE/SOURCE APPLICATION SPECIES anti-DLL1 (human), mAb (D1L165-6) AG-20A-0074 50 μg | 100 μg Mouse IgG1N ELISA, WB Hu anti-DLL1 (mouse), mAb (D1L357-1-4) AG-20A-0085 50 μg | 100 μg Rat IgG2N ELISA, WB Ms anti-DLL1 (mouse), mAb (30B11.1) AG-20B-0053 100 μg Rat IgG2aN FACS, ICC Ms anti-DLL1 (human), pAb AG-25A-0062 100 μg Rabbit ELISA, IHC, WB Hu anti-DLL1 (human), pAb AG-25A-0079 100 μg Rat ELISA, WB Hu

PROTEINS PID SIZE SOURCE ENDOTOXIN SPECIES DLL1 (human) (rec.) AG-40A-0073 10 μg | 50 μg HEK 293 cells <0.1EU/μg Hu DLL1 (human):Fc (human) (rec.) AG-40A-0116Y 10 μg | 50 μg CHO cells <0.01EU/μg Hu DLL1 (mouse):Fc (human) (rec.) AG-40A-0148 10 μg | 50 μg HEK 293 cells <0.1EU/μg Ms

Lanes: M: Marker 1: hDLL1-Fc, 0 min 2: hDLL1-Fc, 10 min 3: hDLL1-Fc, 30 min 4: hDLL1-Fc, 1 h GAPDH (loading control) 5: hDLL1-Fc, 2 h HES1 6: hDLL1-Fc, 4 h 7: hDLL1-Fc, 8 h 8: hDLL1-Fc, 24 h

FIGURE: DLL1 (human):Fc (human) (rec.) (AG-40A-0116Y) induces the Notch target gene HES1 when coated on a plate. METHOD: A mouse preadipocyte cell line, 3T3L1, was stimulated with 1μg/ml of human DLL1:Fc as in indicated time points and each cell lysate was prepared and subjected to Western blot by using an anti-mouse HES1 or anti-mouse GAPDH specific antibody.

4 APPLICATIONS: FACS: Flow Cytometry; FUNC: Functional Application; ICC: Immunocytochemistry; IHC: Immunohistochemistry IP: Immunoprecipitation; WB: Western blot SPECIES: Bv = Bovine; Dg = Dog; Dr = Drosophila; Hu = Human; Mk = Monkey; Ms = Mouse; Pg = Pig; Rt = Rat; Rb = Rabbit; Prm = Primate www.adipogen.com Notch Ligands DLL3 & DLL4 (Delta-like Protein 3 & 4) BULK

ANTIBODIES PID SIZE ISOTYPE APPLICATION SPECIES anti-DLL4 (human), mAb (DL86-3AG) AG-20A-0080 50 μg | 100 μg Mouse IgG1N ELISA, WB Hu anti-DLL4 (mouse), mAb (9A1.5) AG-20B-0054 100 μg Rat IgG1N FACS, ICC Ms

PROTEINS PID SIZE SOURCE ENDOTOXIN SPECIES DLL3 (human) (rec.) AG-40B-0151 10 μg | 3 x 10 μg HEK 293 cells <0.02EU/μg Hu DLL3 (extracellular domain) (mouse):Fc (human) AG-40A-0178 10 μg HEK 293 cells <0.1EU/μg Ms (rec.) DLL4 (human):Fc (human) (rec.) AG-40A-0077Y 10 μg | 50 μg HEK 293 cells <0.01EU/μg Hu DLL4 (mouse):Fc (human) (rec.) AG-40A-0145 10 μg | 50 μg HEK 293 cells <0.1EU/μg Ms

Literature Citations in High Ranking Journals using AdipoGen's DLL4 (human):Fc (human) [PID# AG-40A-0077Y]: 1. Jagged2 acts as a Delta-like Notch ligand during early hematopoietic cell fate decisions: I. Van de Walle, et al.; Blood 117, 4449 (2011) 2. Notch regulates BMP responsiveness and lateral branching in vessel networks via SMAD6: K.P. Mouillesseaux, et al.; Nat. Commun. 7, 13247 (2016)

Notch Ligands Jagged-1 & Jagged-2 BULK

ANTIBODIES PID SIZE ISOTYPE APPLICATION SPECIES anti-Jagged-1 (human), mAb (J1G53-3) AG-20A-0049 100 μg Mouse IgG1N ELISA, FACS, IHC, Hu WB anti-Jagged-1 (human), mAb (J1G53-3) (FITC) AG-20A-0049F 50 μg Mouse IgG1N FACS, WB Hu anti-Jagged-1 (human), mAb (J1G74-7) AG-20A-0050 100 μg Mouse IgG1N ELISA, FACS, WB Hu

PROTEINS PID SIZE SOURCE ENDOTOXIN SPECIES Jagged-1 (human):Fc (human) (rec.) AG-40A-0081 10 μg | 50 μg HEK 293 cells <0.1EU/μg Hu Jagged-1 (mouse):Fc (human) (rec.) AG-40A-0157T 10 μg | 50 μg HEK 293 cells <0.01EU/μg Ms Jagged-2 (human):Fc (human) (rec.) AG-40A-0155Y 10 μg HEK 293 cells <0.1EU/μg Hu Jagged-2 (mouse):Fc (human) (rec.) AG-40A-0183 10 μg | 50 μg HEK 293 cells <0.1EU/μg Ms

6 FIGURE: 4 Induction of IL-6 expression in human dermal fibroblasts by Jagged-1 (human):Fc (human) (rec.) (Prod. No. AG-40A-0081). Fold induction METHOD: Jagged-1 (human):Fc was coated on a 12-well plate at 1μg/ml overnight at 4°C. Human dermal fibroblasts were cultured in the 2 presence or absence of Jagged-1 (human):Fc for 72 hours. Real time quantitative PCR was used to quantify the expression of IL-6. Picture courtesy of the lab of Prof. Gian-Paolo Dotto, Department of Biochemistry, University of Lausanne 0 CTRL Jagged-1

LATEST INSIGHT

New role of Jagged-1 & OX40L in the selective induction of Treg proliferation

P. Kumar, et al. reported that the Notch ligand Jagged-1 and the TNF superfamily ligand OX40L induce selective proliferation of functional regulatory T cells (Tregs) independent of canonical TCR signaling (in the absence of anti-CD3/CD28 activation) when used together as soluble recombinant ligands. This activation of Tregs by Jagged/OX40L works in an IL-2 dependent way without activating effector T cells. This novel “TCR-independent” strategy using Jagged-1, OX40L and IL-2 for the selective expansion of functional Tregs could have therapeutic implications in various autoimmune diseases including T1D. LIT: Soluble OX40L and JAG1 Induce Selective Proliferation of Functional Regulatory T-Cells Independent of canonical TCR signaling: P. Kumar, et al.; Sci. Rep. 7, 39751 (2017)

5 For updated prices and additional information visit www.adipogen.com or contact your local distributor. Non-Classical Notch Ligands

DLK1 & DLK2 (Protein Delta Homolog 1)

ANTIBODIES PID SIZE ISOTYPE/SOURCE APPLICATION SPECIES anti-DLK1 (human), mAb (PF13-3) AG-20A-0069 50 μg | 100 μg Mouse IgG1N ELISA, FACS, IHC, Hu WB anti-DLK1 (human), mAb (PF299-1) AG-20A-0070 50 μg | 100 μg Mouse IgG1N ELISA, FACS, IHC, Hu WB anti-DLK1 (mouse), mAb (PF105B) AG-20A-0057 50 μg | 100 μg Rat IgG2aN ELISA, WB Ms anti-DLK1 (mouse), mAb (PF183E) AG-20A-0058Y 50 μg | 100 μg Rat IgG2aN ELISA, WB Ms anti-DLK1 (human), pAb AG-25A-0091 100 μg Rat ELISA, FACS, WB Hu anti-DLK1 (human), pAb AG-25A-0092 100 μg Rabbit ELISA, IHC, WB Hu

PROTEINS PID SIZE SOURCE ENDOTOXIN SPECIES DLK1 (human) (rec.) AG-40A-0133 10 μg | 50 μg HEK 293 cells <0.1EU/μg Hu DLK1 (human):Fc (human) (rec.) AG-40B-0152 10 μg | 3 x 10 μg HEK 293 cells <0.01EU/μg Hu DLK1 (mouse):Fc (human) (rec.) AG-40A-0107Y 10 μg | 3 x 10 μg HEK 293 cells <0.1EU/μg Ms DLK2 (human):Fc (human) (rec.) AG-40A-0158 10 μg | 50 μg HEK 293 cells <0.1EU/μg Hu

DLK1, Soluble (human) ELISA Kit AG-45A-0032Y 96 wells Species reactivity: Human Sensitivity: 336 pg/ml Range: 0.47 to 30 ng/ml Assay type: Sandwich Sample type: Serum, Cell Culture Supernatant

Non-Confirmed Notch Ligand

DNER (Delta and Notch-like Epidermal Growth Factor-related Receptor)

ANTIBODIES PID SIZE ISOTYPE/SOURCE APPLICATION SPECIES anti-DNER (human), mAb (DR324-4) AG-20A-0078 50 μg | 100 μg Mouse IgG2aN ELISA, WB Hu anti-DNER (human), pAb AG-25A-0102 100 μg Rabbit ELISA, WB Hu

PROTEINS PID SIZE SOURCE ENDOTOXIN SPECIES DNER (extracellular domain) (human) (rec.) AG-40A-0137Y 10 μg | 3 x 10 μg HEK 293 cells <0.1EU/μg Hu DNER (extracellular domain) (human):Fc (human) AG-40A-0119 10 μg | 50 μg HEK 293 cells <0.1EU/μg Hu (rec.) DNER (extracellular domain) (mouse):Fc (human) AG-40A-0177 10 μg | 50 μg HEK 293 cells <0.1EU/μg Ms (rec.)

6 APPLICATIONS: FACS: Flow Cytometry; FUNC: Functional Application; ICC: Immunocytochemistry; IHC: Immunohistochemistry IP: Immunoprecipitation; WB: Western blot SPECIES: Bv = Bovine; Dg = Dog; Dr = Drosophila; Hu = Human; Mk = Monkey; Ms = Mouse; Pg = Pig; Rt = Rat; Rb = Rabbit; Prm = Primate www.adipogen.com Notch Target HES1 (Hairy and Enhancer of Split 1)

Hes genes, encoding basic helix–loop–helix (HLH) transcriptional repressors, are seven members in human, expressed in many tissues and playing various roles mainly in development. Hes1, Hes5, and Hes7 are downstream effectors of canonical Notch signaling. Hes1 plays a crucial role in the control and regulation of cell cycle, proliferation, cell differentiation, survival and apoptosis in neu- ronal, endocrine and T-lymphocyte progenitors as well as various cancers and is a key target gene of the Notch signaling pathway.

ANTIBODY PID SIZE ISOTYPE APPLICATION SPECIES anti-HES1, mAb (7H11) AG-20T-0400 100 μg Mouse IgG2bN ELISA, FACS, IP, WB Hu, Ms, Rt

PROTEIN PID SIZE SOURCE ENDOTOXIN SPECIES HES1 (human) (rec.) (His) AG-40A-0180 10 μg | 50 μg E. coli n.a. Hu

FIGURE (LEFT): Western blot analysis on cell lysates HeLA cells (lane1), HT-29 cell (lane2) and BeWo cell (lane 3) using anti-HES1, mAb (7H11) at 1μg/ml.

FIGURE (RIGHT): Immunoprecipitation analysis on BeWo cell lysate using anti-HES1, mAb (7H11). Lane 1: BeWo cell lysate Lane 2: Precipitation from BeWo cell lysate (400μg) at 2μg Lane 3: Precipitation from BeWo cell lysate (400μg) at 5μg Lane 4: Precipitation from PBS at 5μg

E3 Ubiquitin Ligases (E3UBLs) & Deubiquitinating Enzymes (DUBs)

In contrast to other signaling pathways, where a cascade of second messengers is at stake, the activated Notch receptor is itself transformed into a transcriptional activator, NICD. As a consequence, affecting NICD production and quantity directly affects Notch- dependent response. Therefore, regulating this pathway means controlling spatiotemporal production and maintenance of active receptors and ligands at the cell surface, efficiency of signal transduction and stability of NICD. These key steps all involve ubiquitination events. Dysregulations of these events might be involved in various pathological processes in which the Notch signaling is disrupted. Recently, USP12 has been shown to be important for Notch degradation. The deubiquitinating complex USP12/UAF1 is recruited by Itch to non-activated Notch and regulates Notch trafficking toward lysosomal degradation.

LIT: Ubiquitinations in the Notch signaling pathway: J. Moretti & C. Brou; Int. J. Mol. Sci. 14, 6359 (2013)

KIT PID SIZE SOURCE SPECIES TYPE ITCH (human) Ubiquitination Kit AG-44T-0117 Kit Hu E3UBLs

PROTEINS PID SIZE SOURCE SPECIES TYPE ITCH Isoform 2 (human) (rec.) AG-40T-0284 100 μg E. coli Hu E3UBLs MDM2 (human) (rec.) AG-40T-0289 50 μg E. coli Hu E3UBLs MDM2 (human) (rec.) (GST) AG-40T-0290 50 μg E. coli Hu E3UBLs UAF1 (human) (rec.) (His) AG-40T-0406 50 μg Sf21 cells Hu DUBs USP1/UAF1 Complex (human) (rec.) (His) AG-40T-0536 50 μg Sf21 cells Hu DUBs USP9x Isoform 2 (human) (rec.) (His) AG-40T-0545 25 μg Sf21 cells Hu DUBs USP12 (human) (rec.) (His) AG-40T-0570 50 μg Sf21 cells Hu DUBs USP12/UAF1 Complex (human) (rec.) (His) AG-40T-0571 1 vial Sf21 cells Hu DUBs USP28 (human) (rec.) (His) AG-40T-0541 100 μg Sf21 cells Hu DUBs

7 For updated prices and additional information visit www.adipogen.com or contact your local distributor. www.adipogen.com ADAM17 – Important Sheddase in the Notch Pathway

ADAM17 (Disintegrin and metalloproteinase domain-containing protein 17), also called TACE (Tumor Necrosis Factor-D-Converting Enzyme) is the prototype of the ADAM family of ectodomain shedding proteases (sheddase). ADAM17 is responsible for the processing of a diverse variety of membrane-anchored cytokines, cell adhesion molecules, receptors, ligands and enzymes, including processing of tumor necrosis factor D at the surface of the cell and extracellular Notch Receptor 1. As the proteolytic cleavage is an indispensable activation event for many of these substrates, ADAM17 has emerged as an attractive therapeutic target for the treatment of inflammatory diseases (e.g. rheumatoid arthritis) or inflammation associated cancer. NEW anti-ADAM17 (human), mAb (rec.) (blocking) (D1(A12)) (preservative-free) 50 AG-27B-6000PF 100 μg 40 30 anti-ADAM17 (human), mAb (rec.) (blocking) (D1(A12)) 20 (Fab Fragment) (His) (preservative-free) sTNF- D (pg/ml) 10 0 AG-27B-6003PF 100 μg

No IgG

Recognizes the catalytic and non-catalytic domain of human ADAM17 (TACE) D1(A12) IgG through its variable light (VL) domain and variable heavy (VH) domain, respec- Human plasma IgG tively. Does not bind recombinant mouse ADAM17 ectodomain.

Functional Application (Blocking): Inhibits ADAM17 activity at 15μg/ml (200nM). FIGURE: D1(A12) IgG inhibits constitutive shedding of TNF-D from IGROV1 (human ovarian cancer cell line) into culture medium. Medium was collected after 48 hours of LIT: Cross-domain inhibition of TACE ectodomain: C.J. Tape, et al.; PNAS 108, 5578 (2011) incubation with or without IgGs at 200nM.

Notch Processing / J-Secretase Inhibitors

Compound E DAPT AG-CR1-0081 250 μg | 1 mg | 5 mg AG-CR1-0016 5 mg | 25 mg

Formula: C27H24F2N4O3 Formula: C23H26F2N2O4 MW: 490.5 MW: 432.5 CAS: 209986-17-4 CAS: 208255-80-5 Non-competitive J-secretase inhibitor. Cell permeable J-secretase inhibitor. Notch processing inhibitor. Notch processing inhibitor.

NEW Compound 34 Withaferin A AG-CR1-0007 200 μg | 1 mg AG-CN2-0490 1 mg | 5 mg | 10 mg

Formula: C31H24F3N3O3 CH3 OH Formula: C28H38O6 MW: 543.5 H O O N OH F MW: 470.6 H3C CAS: 564462-36-8 N H CAS: 5119-48-2 CH3 O O N H F O Cell permeable, highly potent inhibitor CH H Notch receptors modulator. 3 of J-secretase (IC50= 0.06nM). F LIT: J. Lee, et al.; Breast Cancer Res. Treat. O 136, 45 (2012) OH

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