Quick viewing(Text Mode)

Toll-Like Receptors

Toll-Like Receptors

Toll-like Receptors

TLR2/TLR6 TLR1/TLR2 TLR5 TLR4 TLR10 CD14 CD14 CD36

CD14 TIRAP TIRAP MD-2 MyD88 TIRAP MyD88 MyD88 TRAM MyD88 MyD88

Endosome TLR7 RIP1 IRAK4 IRAK1/2 TLR8 TLR9 TLR3

TRAF-6 TAB1/2 TAK1 TRAF-6 MyD88 MyD88 TRIF MyD88 TRAF-3 TRAF-3 TANK IKK IRF7 MKK RIP1 TBK1 Proteasome IKKε TAB1/2 IRF7 Homodimer TAK1 TRAF-6 TRAF-3 IκB Proteasome TANK IKK IκB IRF3 NFκB JNK IκB MKK p38 TBK1 IKKε IκB NFκB p38 JNK IRF7 IRF3 Homodimer IRF3

IRF7 Homodimer IRF3 Homodimer Toll-like Receptors

Toll-like receptors are a family of type I transmembrane pattern recognition receptors (PRRs) that sense invading or endogenous damage signals and initiate the innate and adaptive immune response. There are ten functional TLRs in (TLR1–10) and twelve in mice (TLR1−9, 11−13). Various combinations of TLRs are expressed by different subsets of immune and non-immune cell types such as , , dendritic cells, , B cells, T cells, fibroblasts, endothelial cells, and epithelial cells. Of the human TLRs, TLR1, 2, 4, 5, 6, and 10 are expressed on the cell surface and primarily recognize microbial membrane and/or cell wall components, while TLR3, 7, 8, and 9 are expressed in the membranes of endolysosomal compartments and recognize nucleic acids. TLRs have a variable number of ligand- sensing, leucine-rich repeats (LRR) at their N-terminal ends and a cytoplasmic Toll/IL-1 R (TIR) domain. The TIR domain mediates interactions between TLRs and adaptor involved in regulating TLR signaling including MyD88, TRIF, TRAM, and TIRAP/MAL. Signaling pathways activated downstream of these adaptor molecules promote the expression of pro-inflammatory , chemokines, and type I and type III . Although TLRs provide protection against a wide variety of pathogens, inappropriate or unregulated activation of TLR signaling can lead to chronic inflammatory and autoimmune disorders.

TLR2/TLR6 TLR1/TLR2 TLR5 TLR4 TLR10 Toll-like Receptors (TLRs) CD14 CD14 CD36 TLRs (s) Source CD14 TIRAP TIRAP MD-2 MyD88 TLR1/ TIRAP MyD88 MyD88 Triacyl lipopeptides TRAM TLR2 MyD88 MyD88 TRIF Multiple Pathogens Peptidoglycan (PGN) Bacteria TLR7 Porins Bacteria RIP1 IRAK4 IRAK1/2 TLR8 TLR9 TLR3 TLR2 Zymosan Fungi b-Glycan Fungi TRAF-6 TAB1/2 GPI-mucin TAK1 TRIF TRAF-6 MyD88 MyD88 Envelope MyD88 TRAF-3 TRAF-3 TANK TLR2/ Diacyl lipopeptides Bacteria IKK TLR6 IRF7 (LTA) Bacteria MKK RIP1 Double-stranded RNA Viruses TBK1 Proteasome IKKε TAB1/2 TLR3 Synthetic analog IRF7 Homodimer TAK1 TRAF-6 Poly (I:C) of double- TRAF-3 stranded RNA IκB Proteasome TANK Bacteria IKK (LPS) IκB IRF3 NFκB Glycoinositol- JNK IκB Protozoa MKK phospholipids p38 TBK1 IKKε TLR4 IκB Envelope NFκB Viruses glycoproteins p38 Host-derived HMGB1 JNK IRF7 Endogenous and HSPs IRF3 Homodimer IRF3 TLR5 Bacteria IRF7 Homodimer IRF3 Homodimer TLR7 Single-stranded RNA Viruses TLR8 Single-stranded RNA Viruses Bacteria Unmethylated CpG Protozoa Domain Key TLR9 DNA Leucine-Rich Repeat Viruses Domain (LRR) IRF3 NFκB AP-1 IRF7 NFκB AP-1 IRF3 or IRF7 Mitochondrial DNA Endogenous Toll/IL-1 Domain (TIR) Pro-inflammatory Cytokines: TLR10 Unknown Unknown Domain (DD) IL-1β, TNF-α, IL-6, IL-8, IL-18 Kinase Domain Type I or Type III IFN & IFN-inducible © 2012 R&D Systems, Inc.

For more information, please visit | rndsystems.com/TLR Recombinant TLR Proteins from R&D Systems

R&D Systems offers the widest selection of recombinant human and mouse TLR proteins for investigating the ligand-binding and biochemical properties of different toll-like receptors. Stringent production and purification standards ensure that R&D Systems® proteins will provide researchers with industry-leading bioactivity and lot-to-lot consistency.

TLR3 (µg/mL) 1. 0.001 0.01 0.1 1 10 100 1. ) 1 1 IL-8 Secretion (Mean O.D. ) ) 1. 450/540

450/540 0. 0. 1 450/540 Poly (I:C) 1 0.6 TLR 0.6

0. 0. 0. 0. 450/540

0. 0. (O.D. IL-8 Secretion 0. TNF- (O.D. α Secretion ackground IL-8 Secretion (Mean O.D. Secretion IL-8 ackground 0 0 ) 0 0. 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 0.01 0.1 1 10 100 1000 TLR4/MD2 (µg/mL) Poly (I:C) (µg/mL) TLR-5 (ng/mL)

Recombinant Human TLR4/MD-2 Complex Blocks Recombinant Human TLR3 Inhibits Poly (I:C)- Recombinant Mouse TLR5 Blocks Flagellin- LPS-Induced TNF-α Secretion by PMA- Induced IL-8 Secretion by TLR3-transfected Induced IL-8 Secretion by HT-29 Cells. The HT-29 Differentiated U937 Cells. The U937 human HEK293 Cells. The HEK293 human embryonic human colon adenocarcinoma cell line was treated histiocytic lymphoma cell line was differentiated kidney cell line was transfected with TLR3 and with Flagellin and the indicated concentrations of with PMA and treated with 10 ng/mL treated with increasing concentrations of Poly (I:C) Recombinant Mouse TLR5 Fc Chimera lipopolysaccharide (LPS) that had been pre- (Tocris, Catalog # 4287). CXCL8/IL-8 secretion was (R&D Systems, Catalog # 7915-TR). CXCL8/IL-8 incubated for 1 hour with the indicated measured using the Human CXCL8/IL-8 Quantikine® secretion was measured using the Human CXCL8/ concentrations of Recombinant Human TLR4/MD-2 ELISA Kit (R&D Systems, Catalog # D8000C; orange IL-8 Quantikine® ELISA Kit (R&D Systems, Catalog #

Complex (R&D Systems, Catalog # 3146-TM). line). The stimulatory effect induced by 10 µg/mL of D8000C). The ED50 for this effect is typically Following 20 hours of incubation with the LPS- poly (I:C) was inhibited by treating the cells with 4−24 ng/mL. TLR4/MD-2 complex, TNF-a secretion was increasing concentrations of Recombinant Human measured using the Human TNF-α Quantikine® TLR3 (R&D Systems, Catalog # 1487-TR; blue line).

ELISA Kit (R&D Systems, Catalog # DTA00C). The ED50 for this effect is typically 5-10 µg/mL.

Recombinant TLR Proteins Molecule Species Source Catalog # Human NS0 1484-TR TLR1 Mouse NS0 1476-TR Human NS0 2616-TR TLR2 Mouse Sf21 (baculovirus) 1530-TR Human NS0 1487-TR TLR3 Mouse NS0 3005-TR TLR4 Human NS0 1478-TR TLR4/MD-2 Human NS0 3146-TM TLR5 Mouse CHO 7915-TR Human CHO 7755-TR TLR6 Mouse Sf21 (stably transfected) 1533-TR TLR9 Mouse CHO 7960-TR TLR10 Human CHO 6619-TR TLR11 Mouse CHO 7640-TR TLR12 Mouse CHO 8086-TR

For more information, please visit | rndsystems.com/proteins Tocris® Small Molecules for TLR Research

Tocris is the leading supplier of novel and exclusive tools for life science research. Their portfolio includes a collection of TLR and inhibitors that can be used to characterize the signaling pathways downstream of specific toll-like receptors and determine the effects of TLR signaling on the immune response.

Tocris Small Molecules for TLR Research

Small Molecule Description Catalog # CU-T12-9 Potent TLR1/2 agonist 5414

Pam3CSK4 TLR1/2 agonist 4633

Pam3CSK4 Biotin Biotinylated Pam3CSK4 4636 CU CPT 22 Selective TLR1/2 inhibitor 4884

Pam2CSK4 TLR2/6 agonist 4637

Pam2CSK4 Biotin Biotinylated Pam2CSK4 4638 Poly(I:C) TLR3 agonist 4287 CU CPT 4a Selective TLR3 inhibitor 4883 C34 TLR4 inhibitor 5373 DSR 6434 Potent TLR7 agonist 4809 Imiquimod TLR7 agonist 3700 Resiquimod TLR7 agonist 4536 RWJ 21757 TLR7 agonist 2719 Hydroxychloroquine sulfate TLR9 inhibitor 5648

For more information, please visit | tocris.com

Immunology Literature from Tocris: Immunology Product Listing A collection of over 190 products for immunology research, including research tools for studying chemokine and signaling, chemotaxis, the , immune cell signaling and .

Multiple Sclerosis Poster Multiple sclerosis (MS) is an autoimmune disease characterized by focal demyelination and axon degeneration. Created by Alastair

Wilkins and Richard Ibitoye of University of Bristol, Multiple Sclerosis: from Neurobiology to Therapy Dr R. Ibitoye, Dr A. Wilkins Institute of Clinical Neurosciences, University of Bristol, Learning and Research Building, BS10 5NB, UK. www.tocris.com

Multiple sclerosis (MS) is an autoimmune, acquired mediated neuro-inflammatory disorder characterized by focal demyelination in the central nervous system (CNS) and axonal degeneration. Prevalence varies by region, but in the UK prevalence is between 100 and 300 per 100,000 people. The disease usually presents between the second and sixth decades of life and has a female to male ratio of 2:1. In most there is an episodic (relapsing-remitting) course, with focal neurological symptoms and signs developing over hours to days and improving across weeks to months. With time, the frequency of relapses decreases and there is a tendency to accrue progressive disability. In about 1 in 10 patients the disease is progressive with gradual accrual of disability from onset. Diagnosis is based on a clinical assessment, brain and spinal cord magnetic resonance imaging (MRI) and can be supported by cerebrospinal fluid analysis. There are a broad range of immunomodulatory treatments that reduce relapse rates (Table below), but developing treatments effective in preventing long-term disability remains a challenge.

Neurobiology of Demyelination and Axon Degeneration Products available from Tocris α4β1 Integrins this poster summarizes the neurobiology and current BIO 1211, BIO 5192, LDV FITC α4β1 Integrin α4 integrin receptor is involved in ASIC1 translocation through the blood brain Complement Amiloride, Psalmotoxin 1 barrier and is the target of natalizumab Cytokines ASK1 Glutamate T cell MSC 2032964A, TC ASK 10 Proteases Ca2+-Activated Potassium Channels Autoreactive T cells migrate to the TNF-α Apamin, CyPPA, 1-EBIO, NS 309, TRAM 34, CNS, activate B cells and induce a UCL 1684 ROS (Figure 2) Loss of synaptic local inflammation through transmission Cannabinoid Receptors Redistribution of ion channels ACEA, Arvanil, GP 1a, JWH 133, (R)-(+)- along demyelinated neurons Methanandamide, WIN 55,212-2 CNS inflammation, demyelination and axonal results in an ionic imbalance, Chemokine Receptors degeneration are the pathological hallmarks of MS. Activated microglia which promotes tissue damage7 BMS CCR2 22, BX 471, BX 513, Activation of autoreactive T cells to myelin-related J 113863, RS 102895, RS 504393 + , transition through the blood brain barrier, H Glutamate Complement therapies of MS. ASIC1 receptor Compstatin, NDT 9513727, PMX 205, activation of CNS B cells and microglial activation PMX 53, W 54011 Oligodendrocyte are important steps in these processes. The latter Cytokines and axonal injury releases reactive oxygen species (ROS – Figure 2), AS 101, CRID3, 4-IPP, Pirfenidone, SC 144, which are significant mediators of cellular toxicity. Thalidomide Ca2+ Na+ Ca2+ Na+ Oligodendrocytes are injured in the inflammatory IFNγ milieu resulting in demyelination. Axonal injury occurs Na+ Ca2+ Na+ Andrographolide, Resiquimod early on and progresses over time, disrupting action Immunosuppressants Neuron potential propagation and axon transport. Current Azathioprine, Cyclosporin A, therapies target these processes, reducing FK 506 Mycophenolic acid, Rapamycin, inflammatory activity with therapeutic effect. Inflammation produces Na channel TRPM4 Teriflunomide, Triptolide v Inward Rectifier Potassium (K ) Channels ROS ( ) that cause Ca channel ir mitochondrial injury, which v Gambogic acid, ML 133 in turn promotes -2 Inducible T cell Kinase (ITK) misfolding in the ER7 BMS 509744, CTA 056, PF 06465469 Grey matter Glutamate (Ionotropic) Receptors Axonal injury Lamotrigine isethionate, Riluzole Type 2 Na+/Ca2+ Exchanger hyperintense Axonal injury leads to interrupted Benzamil, Bepridil, KB-R7943, SN-6, lesions axonal transport and accumulation YM 244769 Colocalization of Na+ and Na+/Ca2+ channels of amyloid precursor protein Neural Stem Cells + 17-AAG, INDY, Neuropathiazol, ProINDY, Oligodendrocytes form Na channel is a potential therapeutic target SU 5402 myelin around axons ER Neuronal Metabolism White matter Etifoxine, Methylprednisolone, Figure 2: Important ROS reactions a b L-arginine + 3/2 NADPH + H+ + 2O Increased expression of 2 MOG (35-55), Myelin Basic Protein (87-99), a – Generation of superoxide anion from NADPH-oxidase Nogo-66 (1-40), PLP (139-151) antioxidant such molecular oxygen by NADPH-oxidase 2O 2O . – 2 2 NOS Nrf2 as catalase and superoxide ( adenine dinucleotide phosphate) dismutases in grey matter. DMF, CDDO Im, Curcumin as part of respiratory burst. b – Generation of NADPH NADP+ + H+ Citrulline + 3/2 NADP+ NO ROS nitric oxide by action of nitric oxide synthases SIN-1, SNAP, Spermine NONOate, Tempol Figure 1: MRI scan of a MS brain (NOS), in particular inducible NOS as part of c . RXR Agonists 2O2 T2 weighted axial view of MRI brain scan from a the respiratory burst. c – Detoxification of SOD Catalase Bexarotene, Docosahexaenoic acid, superoxide anion to oxygen and water through Fenretinide, HX 630, Isotretinoin, patient with relapsing remitting MS. Multiple white 2H2O2 2H2O + O2 Retinoic acid matter T2 hyperintense lesions in orientation and actions of superoxide dismutases (SODs) and 4H+ Sphingosine-1-phosphate Receptors distribution typical for MS. peroxidases e.g. catalase. CS 2100, CYM 5442, SEW 2871, Sphingosine-1-phosphate, VPC 23019, W146 STAT3 Drug Targets Colivelin, NSC 74859, Stattic Genes and the Environment Measuring Disease Activity and Disability Tankyrase Ever since the first descriptions of MS by Charcot in 1865, Central to our understanding of an inflammatory basis for MS relapses is the concept of disease activity. MR imaging in its capacity to There are many licensed drug treatments for relapsing-remitting Multiple Sclerosis, JW 55, MN 64, XAV 939 clinicians and scientists have considered the risk factors for demonstrate subclinical CNS inflammation is a powerful tool in the measurement of disease activity (Figure 1). Loss of CNS volume (atrophy), which reduce the burden of relapse. β-1b was the first of these, introduced TRPM4 developing MS. There is evidence of a polygenetic determinant of calculated between interval scans, also correlates with the gradual accrual of disability in progressive disease. in 1993 and is of modest efficacy. More effective agents often with a more 9-Phenanthrol, DIDS risk, in particular specific human leukocyte haplotypes significant adverse event profile have been developed, in particular the monoclonal Vitamin D Measuring disability is essential to assessing potential treatment effects on progressive disease. The Expanded Disability Status Scale (EDSS) Calcitriol, EB 1089 confer higher susceptibility. In addition, environmental factors such although initially described in 19613, remains the de facto standard in clinical trials as a measure of disability. As a primarily motor system . Oral agents of modest efficacy and good tolerability such as dimethyl as exposure to infectious agents may modify this risk. fumarate and teriflunomide, have further expanded our formularies. Voltage-Gated Potassium Channels dependent scale with non-linear ordinal characteristics, EDSS is an insensitive measure of change in disability. The Multiple Sclerosis Functional 4-Aminopyridine, ADWX 1, CP 339818, Epidemiological studies confirm variation in the incidence of MS Composite (MSFC) was developed to improve on this and generates a parametric, continuous variable. MSFC improves on EDSS by formally Despite good progress there remains no significant randomized control trial evidence, Kaliotoxin, Margatoxin within and between countries and a latitudinal gradient with higher assessing cognition, and being more sensitive to change. Other potential surrogates for disability such as optic coherence tomography, showing that any of these therapies have significant impact on long-term disability. Voltage-Gated Sodium Channels risk further from the equator. Latitude alone however fails to are being researched. APETx2, Flecainide acetate, Lidocaine, Drug Target Effect explain intra-regional differences, which may be better explained QX 314 chloride, Tetrodotoxin citrate, Veratridine by ethnicity-related susceptibility and migration patterns. There is The Expanded Disability Status Scale (EDSS) Interferon β Interferon receptor and Pleiotropic – modulates various aspects of Wnt Inhibitors for example a lower incidence of MS in Americans with African STAT signaling pathways 1 Glatiramer Possibly MHC II on antigen Hypothesized induction and activation of Cardionogen 1, FH 535, IWP 2, IWP 4, ancestry compared with those of North European ancestry . Wnt-C59 acetate presenting cells suppressor T cell population Twin studies 2 confirm a genetic determinant of risk, which is Teriflunomide Inhibitor of mitochondrial Reduces uridine synthesis polygenetic. In keeping with an autoimmune basis to disease, References dioorate dehydrogenase Impairs clonal expansion of activated 1. Davenport (1921) Science 54 391 HLA haplotypes, in particular DR2, have been identified as Death 2. Ebers (1982) The Lancet 2 1278 susceptibility factors. EDSS EDSS EDSS EDSS EDSS EDSS EDSS EDSS EDSS EDSS EDSS 3. Kurtzke (1961) Neurology 11 390 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Dimethyl Kelch-like ECH-associated Activates Nrf2 antioxidant response 4. Gold et al (2012) NEJM 367 1098 Epidemiological studies show an association between specific viral 4 5. Polman et al (2006) NEJM 356 899 Normal No disability Minimal Moderate Relatively Disability Assistance Essentially Restricted Bedridden fumarate protein 1 pathway and MS risk. Epstein Barr exposure during an 6. Cohen et al (2012) Lancet 380 1819 neurological with only disability disability severe affects required to restricted to to bed or and unable 5 age-linked period of susceptibility is an emerging hypothesis as an Natalizumab α4 integrin receptor Impairs migration of activated autoreactive 7. Dendrou et al (2015) Nature 15 545 function minimal signs disability full daily walk and wheelchair wheelchair to communicate T cells into CNS environmental risk factor for MS. In addition, low vitamin D levels activities work effectively or For copies of this poster, Alemtuzumab6 CD52 Peripheral B and T cell depletion are linked to risk of relapse, and studies are assessing whether eat/swallow please visit www.tocris.com vitamin D replacement influences MS disease course. Modulates autoreactive immune response © 2016 Tocris Cookson, Ltd. Tocris is a Bio-Techne brand

To download or request a copy please visit | tocris.com/requestliterature Unconjugated & Fluorochrome-conjugated TLR Antibodies

R&D Systems and Novus Biologicals together offer the most comprehensive selection of unconjugated and fluorochrome-conjugated antibodies for detecting toll-like receptors and related molecules using a variety of different applications. Our catalogs include antibodies that are validated for flow cytometry, immunocytochemistry/immunofluorescence, immunohistochemistry, Western blot, and blocking/ neutralization.

Blocking/Neutralization Antibodies for TLRs TLR2 ntibo g/mL TLR4 ntibo g/mL - - - IL-8 Secetion Mean D IL-8 Secetion Mean D . . . . 45/54 . . 45/54 . .

. ipopeptide . . . PS . Antibody . . . . . 45/54 . . 45/54 . . IL-8 Secetion Mean D IL-8 Secetion . . D Mean IL-8 Secetion . . - -

PamCS4 g/mL LPS ng/mL TLR2 Ligand-Induced IL-8 Secretion and TLR4/MD2 Ligand-Induced IL-8 Secretion and Neutralization using an Anti-Human TLR2 Neutralization using an Anti-Human TLR4 Antibody. Antibody. The HEK293 human embryonic kidney The HEK293 human embryonic kidney cell line co- cell line transfected with human TLR2 was treated transfected with human TLR4 and MD-2 was with increasing concentrations of the synthetic tri- treated with increasing concentrations of

palmitoylated lipopeptide Pam3CSK4 (Tocris, lipopolysaccharide (LPS). IL-8 secretion was Catalog # 4633). IL-8 secretion was measured measured using the Human CXCL8/IL-8 Quantikine® using the Human CXCL8/IL-8 Quantikine® ELISA Kit ELISA Kit (R&D Systems, Catalog # D8000C; orange (R&D Systems, Catalog # D8000C; orange line). line). The stimulatory effect induced by 75 ng/mL The stimulatory effect induced by 0.5 µg/mL LPS was neutralized by treating the cells with

Pam3CSK4 was neutralized by treating the cells with increasing concentrations of a Goat Anti-Human increasing concentrations of a Mouse Anti-Human TLR4 Antigen Affinity-purified Polyclonal Antibody TLR2 Monoclonal Antibody (R&D Systems, Catalog (R&D Systems, Catalog # AF1478; blue line). The

# MAB2616; blue line). The ND50 is typically ND50 is typically 1.5–7.5 µg/mL. 0.03–0.15 µg/mL.

For additional information on TLRs and other pattern recognition R&D Systems Tools for Cell Biology ResearchTM Activation Signaling Pathways receptor families, explore R&D Systems pattern recognition receptor- Virus Detection and Induction Signal 2 Signal 1 K+ Efflux Bacterial Type III or of Antiviral Signaling Pathways Pore-forming Toxins IV Secretion Systems Endogenous K+ Channel Cytokine Particulate Activators by RIG-I-like Receptors ATP RNA or DNA virus P2X7 Receptor Pannexin-1 Microbial K+ Ligand IL-1 RAcP TM Ca2+ related signaling pathways at rndsystems.com/Pathways/Immunology IL-1 RI PAMP ROS P P P Ca2+ Channel TIRAP MyD88 ROS AT-rich dsDNA 5'-ppp-ssRNA Long dsRNA Toll-like Phagolysosome 2+ Receptor MyD88 Ca Influx RNA Pol III R&D Systems Tools for Cell Biology Research R&D Systems Tools for Cell Biology ResearchTM Short dsRNA TRAM Membrane P P P LGP2 TRIF NLRP3 Perturbations NOD-like Receptor MKK 5'-ppp-dsRNA or or NLRP3 Inflammasome Bacterial Type III or IV IKK ASC Signaling Pathways Secretion Systems Cardinal RIG-I MDA5R&D Systems Tools for Cell Biology Research + Channel 2+ Channel Pannexin-1 K Ca 2+ -Inflammasome or Damage-Activated TRIM25 and Riplet E1, E2 + Efflux Particulate Ub Ub Ub Ub K Ca Oligomerization E3 Ub Ligases P2X7 Receptor Activators K63-linked Ub Chains Toll-like ATP JNK TM Pro--1 Pro-IL-1β Ub Ub Ub Ub Receptor PAMP C-Type Lectin Receptor Active Caspase-1 K63-linked Ub Chains K+ p38 Bacterial Type III or IV Ca2+ Influx Secretion Systems Ub Ub Ub Ub Membrane Perturbations IκB Signaling PathwaysPro-IL-18 Mature IL-1β Ub Ub Ub Ub Ub Ub Ub Ub ROS Phagosome NF-κB ROS K63-linked poly-Ub RIG-I K63-linked poly-Ub MDA5 Toll-like Receptor NLRC3 or NLRX1 Phagolysosome incle Dectin CytokineDA Processing IκB Dectin Phagosome Transcription of Pro-IL-1β and Pro-IL-18 NLRP7/NALP7 or FcRγ γ DIR • Nod-like Receptor Signaling • Toll-like Receptor Signaling or Endosome DSIN FcR FcRγ Mature IL-18 IPS-1/MAVS/VISA NLRP12/NALP12 Signaling Pathways Inflammasome Activation NLRC4/IPAF I/ TRADD NF-κB AP-1 ) Bacterial PGN Components NLRP3/NALP3 STranscription ofS A ly TRAF-3 Src on TRAF-2 IL-1β & IL-18 Src S n TLR1/TLR2 NLRP1/NALP1 tio TRAF-6 TLR4 Proteasome S va Ras Active IL-1β ti TOM70 SP SR SP or c a HSP90 RS SP Active IL-18SP or -I FADD RIP1 TLR2/TLR6 N IG SP R Ras ( NOD2 MD-2 Inflammasome Activation Signaling Pathways Signaling Activation Inflammasome NI CD14 NOD1 R Pro-Caspase-8 CD14 TLR5 RS RS Pro-Caspase-10 CD14 Pathways Pathways AR R Signaling TRAF-3 TLR10 TANK CD36 TRAM TIRAP TIRAP NLRC4 Raf NAIP IPAF Pγ SP or NLRP7 or Domain Key © 2012 R&D Systems, Inc. TRIF MyD88 MyD88 cIAP-1 or NLRP12 Iα SP MyD88 TIRAP cIAP-1 or cIAP-2 cIAP-2 NLRP3/ Toll-Like Receptor Domains Inflammasome Domains N TBK1 IKK epsilon IKK CARD9 NALP3 RhoA PIP RIP2/RICK Toll/IL-1 Receptor Domain (TIR) NACHT-Associated DomainPγ (NAD) DIR MyD88 RIP2/RICK Pro-Caspase-1 Death Domain (DD) Nucleotide-binding/Oligomerization Domain (NACHT)Endosome ASC DA R P P MyD88 or Toll-like Receptor/Nod-like Receptor Domain Pyrin Domain R IRF3 IRF7 Proteasome RIP1 Pro-Caspase-5 IP Ub Ub Ub Ub Cardinal Leucine-Rich Repeat Domain (LRR) Caspase Recruitment Domain (CARD) I kappa B TAK1 ASC Pro-Caspase-1 P TAB1/2/3 NLRC4 NF I I kappa B P NLRP1/ NFκ p Rel Function to Find Domain (FIIND) IKK IRF3 or IRF7 IPAF mRNA Decay Pδ IRAK4 Endosome TLR7 NALP1 yD Endoplasmic NF-kappa B IRAK1/2 Pro-Caspase-1 yD Reticulum IRF3 IRF7 TLR8 STING Homodimer Homodimer TAB1/2 Inflammasome IP TAK1 Oligomerization ASC TRAF-3 TR TLR9 Proteasome ARD AF-6 TLR3 I κ B Raf cl TANK TRAF-6 • Inflammasome Activation Pathways www.RnDSystems.com/Pathways_InflammasomeActivationA ASC Pro-Caspase-1 MKK TRAF-3 P Pro-Caspase-1 IKK I κ B alcineurin IRF3 or IRF7 TBK1 NF-kappa B MyD88 NFκB I Phosphatase MyD88 JNK Type I or Type III IFN & Pro-inflammatoryIKKε Cytokines: IFN-inducible genes IL-1 beta, IL-6, IL-8,Pr oTNF-alphat ERK1/2 Pyroptosis easome MKK TRIF p38 Active Caspase-1 almodulin IRF7 MyD88 For research use only. Not for use in diagnostic procedures. Proteasome Domain Key

NOD-like Receptor Signaling Pathways Signaling Receptor NOD-like Mature-IL-1β Iκ Caspase Recruitment Domain (CARD) Repressor Domain Death Effector Domain (DED) alcineurin RNA Domain Death Domain (DD) Kinase Domain IRF3 or IRF7 Mature IL-18 NFκ p Rel © 2014 R&D Systems Homodimer Pro-IL-1β IκB Phosphatase IRF7 Homodimer Pro-IL-18 Iκ IRF3 RIP1 NFκ TAB1/2 almodulin Receptors RIG-I-like by and Induction of Antiviral Signaling Pathways Virus Detection IκB TAK1 p R/ rndsystems.com/Pathways_RIG-I TR N Toll-like Receptor Signaling Pathways NF-κB Proteasome AF-6 Inflammatory Response NFA TRAF-3 For research use only. Not for use in diagnostic procedures. JNK IKK Domain Key TANK • Pathogen or Damage-activated Caspase Recruitment Domain (CARD) a p38 κ Pyrin Domain (PYD) I B IRF3 or IRF7 Baculovirus Inhibitor of Repeat Domain (BIR) IP Receptor Type I IFN & Unknown N-terminal Domain Nucleotide-binding/Oligomerization Domain (NACHT or NOD) NFκ p Rel AP MKK IFN-inducible genes Leucine-Rich Repeat Domain (LRR) IRF3 Homodimer IκB NF-κB TBK1 AP-1 NACHT-Associated Signaling Pathways Receptor Lectin C-Type or Damage-Activated Pathogen- Domain (NAD) ε Function to Find Domain (FIIND) © 2013 R&D Systems, Inc. IKK NFκB NFκ p NFκ p p38 Pro-inflammatoryIL-18 Cytokines: Rel P NFκ p NFκ p Rel NFA IL-1β TNF-α Inactive JNK C-Type Lectin Receptor Signaling Oligomerized inflammasome complexes may consist of more subunits than what is shown p IL-6 IL-8 IRF3 IRF7 Domain K ey Proinflammatory ytoines Leucine IRF3 Homodimer D -Rich Repea IRF7 Homodimer Domain ey omain (LRR) t

Immunoreceptor yrosineased Activation otif IA arohydrate Recognition Domain RD/type ectinlie Domain D Toll/IL-1 Rec IRF3 RnDSystems.com/Pathways_NLRSignaling Immunoreceptor yrosineased Inhiitory otif II aspase Recruitment Domain ARD Domain ( eptor D TIR) NF-κB eath Domain (DD AP-1 Kinase D ) RnDSystems.com/Pathways_CLRSignaling omain IRF7 Pathways For research use only. Not for use in diagnostic procedures. κ NF- B Proinflamma AP-1 IL -1β, TNF-α, tILory Cytok IRF3 or IRF7 For research use only. Not for use in diagnostic procedures. Type I or -6, IL ines: Type III IFN & IFN-inducible-8, IL-18 genes www.RnDSystems.com/Pathways_TLRSignaling For research use only. Not for use in diagnostic procedures. © 2012 R&D S ystems , Inc. • RIG-I-like Receptor Signaling Pathways

® Infl ammasomes: Intracellular Regulators of Pathogen Recognition, Host Defense, and Infl ammation Mechanisms to Reduce IL-1β or IL-18 Signaling P IL-1 & IL-18 Signaling Pathways Infl ammasome-mediated Caspase-1 Activation Regulates the Secretion of IL-1β & IL-18 IκB Printed copies of R&D Systems pathways or our pattern recognition Inhibitors Mechanisms of Action Nod-like receptors (NLRs) are a subset of pattern recognition receptors (PRRs) found in the cytosol that are essential for detecting invading pathogens and initiating the innate immune response. IL-18 BP IL-18 binding protein (IL-18 BP); Binds to IL-18 with higher a nity than either the cell bound or soluble NLRs are activated either by bacterial, fungal, or viral molecules that contain pathogen-associated molecular patterns (PAMPs), or by nonmicrobial danger signals (DAMPs) released from damaged IKK forms of IL-18 R and prevents IL-18 signaling IL-1ra An IL-1 receptor antagonist that prevents IL-1 from binding to its receptor cells. Upon activation, some NLRs oligomerize to form multiprotein in ammasome complexes that serve as platforms for the recruitment, cleavage, and activation of in ammatory . P IL-1 RII A decoy IL-1 receptor with a short cytoplasmic domain that is incapable of transducing an IL-1 signal In ammasome oligomerization requires two signals, a priming signal that results in the transcription of Pro-IL-1β and Pro-IL-18 (–-—), and a second signal that promotes indirect IκB IL-1β Signaling SIGIRR Single immunoglobulin IL-1-related receptor (SIGIRR); Prevents IL-1 RI/IL-1 RAcP heterodimerization activation of the in ammasome, such as ion or membrane perturbations, reactive oxygen species (ROS), or ATP (˜). In ammasome oligomerization leads to the activation of Caspase-1, followed NFκB . activation Soluble IL-1 RI or RII Soluble receptors that can bind to IL-1 and IL-1 RAcP, but are incapable of propagating a signal TAB1/2 by the maturation and secretion of IL-1β and IL-18 (™-›), and in some cases, an in ammatory form of known as pyroptosis. In ammasome/Caspase-1-dependent secretion of IL-1NADβ FIND CARD . Recruitment of in ammatory cells A soluble receptor that can bind to IL-1/IL-1 RI, but is incapable of propagating a signal; Enhances IL-1 NAD FIND CARD TAK1 TRAF-6 . Induction of secondary cytokines, enzymes, Soluble IL-1 RAcP binding to soluble IL-1 RII and IL-18 stimulates the in ammatory response by inducing the expression of secondary mediators that promote the recruitment of immune cells to the site of the (œ). In addition, IL-18 in ammatory mediators, and acute phase proteins that regulate the local and systemic response enhances the cytolytic activity ofx naturalAD killerNACHT (NK) cells and promotes IFN-γ production. To date, four in ammasome complexes (NLRP1, NLRP3, IPAF, and AIM2) have been partially characterized.NADx ADFIND NACHTCARD IL-18 Rβ NAD FIND CARD IL-1 RI These complexes contain a speci c NLR family protein or AIM2, the ASC and/or Cardinal adaptor proteins, and Pro-Caspase-1. JNK IL-18  IL-18 Signaling x PYD BIR AD NACHT x PYDAD BIRNACHT TRAF-6 Although the secretion of IL-1β and IL-18 are intended to combat infection, constitutive in ammasome activation and the subsequent overproduction of IL-1β or IL-18 can have detrimental e ects P IL-1β  IL-18 Rα receptor-related miniposters listed below can be requested at P MyD88 IL-1 RAcP IL-18 BP that are associated with autoin ammatory and autoimmune disorders. For these reasons, mechanisms that inhibit IL-1β and IL-18 signaling are of interest. Decoy or soluble receptors that sequesterNAD FIND CARD MKK PYD BIRNAD FIND CARD κ IRAK1/2 IRAK4 IL-1β, non-signaling IL-1β antagonists, and disruption of IL-1 receptor heterodimerization are intrinsic pathways that inhibit IL-1β signaling. Similarly, naturally occurring IL-18 bindingPYD proteinBIR NF B AP-1 IL-1 RAcP NAD  x NAD FIND CARD x FIND CARD (IL-18 BP) can prevent IL-18 from bindingAD toNACHT its receptor. Further research is necessary to characterize how in ammasome complexes are activated, the mechanisms by which IL-1β and IL-18 AD NACHT p38 signaling can be regulated, and both the bene cial and detrimental e ects associated with the in ammasome pathway. These  ndings may have therapeutic implications for in ammasome- œ IL-1 RI IL-1 Signaling x AD NACHT x AD NACHT IL-1ra related disorders, including autoin PYD ammatoryBIR disorders, Crohn’s disease, vitiligo, gout, asbestosis, and Alzheimer’s disease.  PYD BIR Inflammatory PYD BIR Response IL-1β IL-1 RAcP NADPYD BIR DOMAIN ORGANIZATIONNAD FIND CARD OF THE NLR PROTEINS OLIGOMERIZED INFLAMMASOME COMPLEXES FIND CARD IL-18 Signaling Active IL-1β IL-1 RII  Human Name Mouse Name Family CARD-containing NLRs NLRP1 (NALP1) Infl ammasome Select Microbial Activators IL-18 Rα IL-1β x AD NACHT Pro-Caspase-1 Pro-Caspase-5 x NADAD FINDNACHTCARD . Induction of IFN-γ in T cells and NK cells Decoy   CIITA (NLRA) CIIta (Nlra) NAD FIND CARDNLRA NLRP1/ASC/Pro-Caspase-1/Pro-Caspase-5 NAD FIND CARD . Induction of secondary pro-in ammatory NAD FIND CARD NAD FIND CARD cytokines, chemokines, cell adhesion molecules IL-18 SIGIRR NAD FIND CARD NAD FIND CARD x NAD BIR FIND CARD NAD FIND CARD . Induction of , NO synthesis IL-18 Rβ NOD1 (NLRC1) Nod1 (Nlrc1)PYDNADAD FINDNACHTCARDNLRC Pro-Caspase-5 Pro-Caspase-1 Pro-Caspase-1 Pro-Caspase-5 x ADBIR NACHT x AD NACHT ASC NLRP1  Bacillus anthracis lethal toxin PYDx AD NACHT Active IL-18 IL-1 RI rndsystems.com x NADAD NACHT  Muramyl dipeptide (MDP) x NAD FIND CARD FIND CARD NOD2 (NLRC2) Nod2 (Nlrc2)x AD NACHT NLRC x AD NACHT Soluble ADBIR NACHT x AD NACHT PYD ASC NLRP1 IPAF (NLRC4) Ipaf (Nlrc4)PYDx BIR NLRC Pro-Caspase-5 Pro-Caspase-1 ASC NLRP1 PYD BIR IL-1 RI or RII PYD ADBIR NACHT Pro-Caspase-1 PYDx ADBIR NACHT PYD BIR PYD BIR Human Name Mouse NamePYD BIR Family BIR-containing NLRs Pro-Caspase-1 Pro-Caspase-5 PYD BIR IL-1 RAcP Pro-Caspase-1 NLRP3 (NALP3) Infl ammasome Select Microbial Activators PYD BIR Signal 2 PYD BIR ASC NLRP1 K+ DAMP/PAMP IL-1 RI NAIP Naip1-7 NLRB Pro-Caspase-1NLRP3/ASC/Cardinal/Pro-Caspase-1Pro-Caspase-1 Pro-Caspase-5 PYD BIR Inflammasome Activation K+ Soluble Cardinal ASC NLRP3 ˜ Bacterial Type III or Human Name Mouse Name Family PYD-containingPro-Caspase-5 NLRsPro-Caspase-1 ASC NLRP1  Adenovirus, Bacterial RNA ATP IL-1 RAcP CardinalPro-Caspase-1ASC NLRP3  / IV Secretion Systems + Pore-forming NLRP1 (NALP1) Nlrp1a-c (Nalp1) NLRP Pro-Caspase-5 Pro-Caspase-1 ASC NLRP1  Danger signals: ATP, NAD , β-amyloid › Pattern Recognition Receptors and the Innate ImmuneCardinal Response Toxins ASC NLRP1 ASC NLRP3 and particulates such as calcium pyrophosphate Signal 1 Secretion of Nlrp2, Nlrp3 (Nalp3), Nlrp4a-g Pro-Caspase-1 P2X Receptor Pannexin-1 Particulate Cardinal ASC NLRP3 Pro-Caspase-1 dihydrate and monosodium urate 7 IL-1β & IL-18 NLRP2-9 (NALP2-9), (Nalp4a-g), Nlrp5, Nlrp6, Pro-Caspase-1 Activators NLRP ASC NLRP1  Xenogenous compounds: Silica, asbestos, and alum – NLRP11-14 (NALP11-14) Nlrp9a-c (Nalp9a-c), Nlrp12Infect ed Tissue Pro-Caspase-1Pro-Caspase-1 Pro-Caspase-5 (Nalp12), Nlrp14 (Nalp14) Pro-Caspase-1  In uenza virus Endogenous Cytokine Active IL-1β Pro-Caspase-1 Active IL-18 NK Cell Inflammation IPAF IPAF  Listeria monocytogenes PAMP NLRP10 (NALP10) Nlrp10 (Nalp10) NLRP Pro-Caspase-1Pro-Caspase-1 CardinalPro-Caspase-1ASC NLRP3Pro-Caspase-5 Microbial Ligand Pro-Caspase-5 Pro-Caspase-1 Pro-Caspase-1ASC NLRP1Pro-Caspase-5  Lipopolysaccharide (LPS) Human Name Mouse Name FamilyCardinalUnknownASC N-terminalNLRP3 Domain Pro-Caspase-1 Pro-Caspase-5  Muramyl dipeptide NAD(MDP) FIND CARD ROS NAD FIND CARD Pro-Caspase-1 IPAF CardinalPro-Caspase-1Pro-Caspase-1ASC NLRPPro-Caspase-53 NAD FIND CARD NLRC3NAD (NOD3), FINDNLRC5CARD IPAF Pro-Caspase-1IPAF IPAF Pro-Caspase-5  Sendai virus IL-1 RAcP Phagosome Nlrc3, Nlrc5 NLRC Cardinal ASC NLRP3 (NOD27) ASC Pro-Caspase-5ASC NLRP1Pro-Caspase-1 ASC NLRP1  Staphylococcus aureus etectio o icroil Leukocyte Recruitmenx AD NACHTt Pro-Caspase-5 Pro-Caspase-1 Pro-Caspase-1Pro-Caspase-1ASC Pro-Caspase-1NLRPPro-Caspase-51 x x Pro-Caspase-1Pro-Caspase-5PathogenPro-Caspase-1NAIP RecognitionPro-Caspase-1 x AD NACHT ROS NLRX1 (NOD9)AD NACHT Nlrx1 NLRX Pro-Caspase-5IPAF Pro-Caspase-1 Pro-Caspase-1ASC NAIPNLRP1 AD NACHT IL-1 RI TIR Domain Pro-Caspase-5 Pro-Caspase-1 ASC IPAF NLRP1 toe tter Pro-Caspase-1 IPAF (NLRC4) InflASC ammasomePro-Caspase-1NLRP1 Select Microbial Activators Death Domain BIRDOMAIN ORGANIZATION OF RELATEDASC ADAPASCTOR PROTEINSPro-Caspase-1NLRP1 PYD Pro-Caspase-5ASC NLRP1Pro-Caspase-1 Pro-Caspase-1 CYTOPLASMIC RECEPTORS Phagolysosome PYD BIR Pro-Caspase-1ASC IPAFNLRP1 IPAF/ASC/Pro-Caspase-1Pro-Caspase-1ASC IPAFNLRP1 PYD BIR ASC NAIP IPAFNLRP1 BIR Toll-like Receptor MyD88 Recoitio Receptor ASC: Apoptosis-associated speck-like protein containing a CARD ASC NLRP1 CardinalNODPro-Caspase-1ASC-LikNAIPe ReceptorsNLRP3 (NLR) & RIG-I-LikePYD Receptors (RLR) TIRAP Pro-Caspase-1 Pro-Caspase-1Pro-Caspase-1 IPAF Cardinal Pro-Caspase-1Pro-Caspase-1ASC NLRP3IPAF IPAF MyD88 Cardinal: CARD inhibitor of NFκB-activating ligands (found only in ) ASC NLRPIPAF 1 Pro-Caspase-1 IPAF  + TRAM Membrane Perturbations Cytokine Secretion Pro-Caspase-1Pro-Caspase-1 Pro-Caspase-1 K 2+ Pro-Caspase-1IPAF Cardinal ASC NLRP3 PatternPattern recognition receptors (PRRs) play a ccriti-riti- MEMBRANE-ASSOCIATED RECEPTORS ASCPro-Caspase-1Pro-Caspase-1  typhimurium Ca TRIF ™ Mature IL-1β IPAF Cardinal ASCPro-Caspase-1TLR ActivationNLRP3 P2X Receptor Viral dsRNA 2+ Cardinal ASC CardinalPro-Caspase-1ASCPro-Caspase-1NLRP3IPAF 7 Shigella exneri Toll-Like Receptors (TLR) Pro-Caspase-1ASC NLRPNAIP3 Cardinal ASC IPAF NLRP3 5'-triphosphate RNA Inflammasome Ca cal role in the detection of invading pathogens DOMAIN KEY Cardinal ASCASCPro-Caspase-1NLRP3 Cardinal ASC NAIPNLRP3 NLRP3 In ammasome Cardinal ASC NLRP3 Pannexin-1 P P P Oligomerization Protein-Protein Interacting Domains Cardinal Pro-Caspase-1ASC NLRP3 Mature IL-18 and subsequent activation of the innate immune Cardinal ASC NLRPNAIP3 Cardinal ASCASC NLRP3 Pyroptosis Viral Envelope ASC Pro-Caspase-1NAIP ATP MKK Viral Envelope Bacterial Triacyl Caspase Recruitment Domain (CARD) Pro-Caspase-1 IPAF Pro-Caspase-1IPAF • Pattern Recognition Receptors & the Innate Immune Response Bacterial LPS Glycoproteins MacrophageCardinal ASC Pro-Caspase-1NLRPIPAF3 response. This response provides the rst line of Glycoproteins Lipopeptides Pro-Caspase-1 IPAF/NAIP/Pro-Caspase-1Pro-Caspase-1 š Pyrin Domain (PYD) ASCAIM2 Pro-Caspase-1Pro-Caspase-1 Helicase Fungal Zymosan Pro-Caspase-1Pro-Caspase-1 ASCPro-Caspase-1Pro-Caspase-1 Protozoan Pro-Caspase-1Pro-Caspase-1 IPAF IPAF Cytokine Processing defense against infectious disease and is primar- & β-Glycan Pro-Caspase-1Pro-Caspase-1IPAF IKK Pro-Caspase-1 2+ GPI-mucin Baculovirus Inhibitor of Apoptosis Repeat Domain (BIR) IPAF Pro-Caspase-1 IPAF RIG-I Ca IPAFIPAF Pro-Caspase-1Pro-Caspase-1IPAFIPAF MDA5 Protozoan Glycoinositol- Unknown N-terminal Domain ASCASCAIM2 Pro-Caspase-1 IPAF  Legionella pneumophila JNK ily mediated by phagocytic, antigen-presenting phospholipids ASC IPAFIPAF Pro-Caspase-1ASCAIM2 IPAFIPAF Bacterial LTA, PGN, LPS, Porins IPAF Pro-Caspase-1 Pro-Caspase-5 IPAF CARD RNA Binding Nucleotide-Binding/Oligomerization Domain Pro-Caspase-1 NAIPIPAFIPAF Pro-Caspase-1 Pro-Caspase-5 IPAF Pro-IL-18 cells such as macrophages and dendritic cells. Pro-Caspase-1 Pro-Caspase-1 NAIPIPAF TLR1 Pro-Caspase-1ASC IPAFIPAF Pro-Caspase-1 Phagosome TLR4 NAIP, CIITA, HET-E, TP-1 Domain (NACHT)ASCPro-Caspase-1 IPAFIPAF Type III or IV Secretion Systems Pro-Caspase-1ASC IPAFIPAFIPAF Pro-Caspase-1 IPAF ASCPro-Caspase-1AIM2 IPAFIPAF or Endosome p38 Pro-IL-1β PRRs expressed by these cells, including mem- Pro-Caspase-5 Pro-Caspase-1 ASCPro-Caspase-1 IPAF Pro-Caspase-1 IPAF Active Caspase-1 LRR Domain Microbial Ligand Recognition Domain ASC NAIP ASC NLRPAIM21 Infl ammasome IPAF Select Microbial Activators TRAF-3 Proteasome Bacterial Diacyl Pro-Caspase-5 Pro-Caspase-1 ASCPro-Caspase-1Pro-Caspase-1IPAF ASC NLRP1 ASC NAIP brane-associated Toll-like receptors (TLRs), and Lipopeptides Leucine-Rich Repeat Domain (LRR) ASCPro-Caspase-1ASCAIM2 NAIPIPAF Pro-Caspase-1AIM2/ASC/Pro-Caspase-1Pro-Caspase-1NAIP IPS-1 TRAF-6 P TLR2 NAIP Pro-Caspase-1 IPAF Hematopoietic Interferon-inducible Nuclear Protein with aNAIP 200 a.a. repeat (HIN200) Bacterial PAMP- NAIPIPAF MD2 NAIP NAIP TRADD P cytoplasmic Nod-like receptors (NLRs) and reti- TLR6 ASC NLRP1 ASC ASCAIM2 containing ComponentsPro-Caspase-1NAIP CD14 Other Domains Bacterial FlagellinASC NLRP1 Pro-Caspase-1 IPAF Pro-Caspase-1 IκB CD14 Pro-Caspase-1 NAIP Pro-Caspase-1 IκB TIR Domain Pro-Caspase-1 IPAFIPAF Pro-Caspase-1ASCAIM2 NAIP  DS-DNA from virus, bacteria, or the host itself AP-1 noic acid inducible I-like helicase receptors CD14 NACHT-Associated Domain (NAD) Pro-Caspase-1ASC IPAFIPAF IPAF Pro-Caspase-1 Pro-Caspase-1 IPAF Pro-Caspase-1 IPAFIPAF NFκB TLR5 FunctionPro-Caspase-1 to Find Domain (FIIND) IPAF Pro-Caspase-1ASCAIM2 NALP3 NFκB (RLRs), recognize speci c, conserved pathogen- TRAM TIRAP CD36 Pro-Caspase-1 Pro-Caspase-1 IPAFIPAF Transcription of Death Domain TIRAP UnknowActivationn Domain (AD) Pro-Caspase-1 Pro-Caspase-1ASC IPAF TRIF MyD88 ASC CardinalIPAF ASC NLRP3 NACHT IPAF IL-1β and IL-18 associassociateated molecular patterns (PAMPs) that are MyD88 ASC Cardinal ASC NLRPOligomerized3 Pro-Caspase-1in ammasome complexes may consist of more subunitsNALP1 than what is shown. Cardinal ASC NLRP3 ASC IPAF TIRAP TLR10 Cardinal ASC NLRP3 Pro-Caspase-1ASCASCAIM2 PYRPro-Caspase-1NAD LRR FINDCARD — present in microbial proteins, nucleic acids, lip- This illustration represents general processes suggested in the scienti c literature and is Pro-Caspase-1notASC to be considered comprehensive nor de nitive. 2010,ASC 2015 R&D Systems Pro-in ammatory Cytokines MyD88 Pro-Caspase-1 Bacterial ASC Monomeric Flagellin MP_In ammasomes_1550 Pro-Caspase-1ASC PGN ASC ids, and carbohydrates. These PAMP-containing Pro-Caspase-1 Pro-Caspase-1 ASC TRAF-3 Pro-Caspase-1 ASCAIM2 ComponentsPro-Caspase-1 Pro-Caspase-1ASC IPAF MyD88 Pro-Caspase-1 ASCAIM2 Pro-Caspase-1ASCAIM2 molecules act as ligands to trigger PRR-depen- TANK Global [email protected] [email protected] Pro-Caspase-1 FADD TRAF-3 ASCAIM2 Pro-Caspase-1ASC RIP1 orth Aerica TELASC 800AIM2 343 7475 Pro-Caspase-1 TRAF-6 dent intracellular signaling pathways that ulti- IPAF IPAFMeso-DAP TANK • : Intracellular Regulators of Pathogen Recognition, Kinase Domain MyD88 IPAFurope iddle ast Africa TEL +44 (0)1235IPAF 529449 ASCAIM2 RIP1 LEARN MORE ASCAIM2 Pro-Caspase-1 hina [email protected] TEL +86 (21) 52380373 ASCMDPAIM2 TAK1 rndsystems.com/ mately induce the expression of pro-in amma- TAB1/2 IRAK4 IRAK1/2 ASCAIM2 Pro-Caspase-1 IPAFRest of orld bio-techne.com/fiPro-Caspase-1 nd-us/distributors TEL +1 612 379ASC 2956AIM2 Inflammasome inflammasome TAK1 Kinase Domain Pro-Caspase-1 CARD NAD IPAF ASCAIM2 bio-techne.com tory and antiviral cytokines. Secretion of these TRAF-6 Death Domain IPAF Pro-Caspase-1 IPAF TBK1 IKKε P P ASC Oligomerization NALP3, NALP1, ASC NOD1 ASCAIM2 cytokines at the site of an infection promotes the NAIP or IPAF NACHT LRRNAIP NAIP RIP2 NAIP TBK1 IKKε recruitment of neutrophils and natural killer TRAF-6 Unmethylated CpG DNA cIAP-1 or -2 (NK) cells, which eliminate pathogenic microbes IKK Pro-Caspase-1 IPAF CARD9 Caspase-8, Endosome Viral ssRNPro-Caspase-1A IPAF -10 TRAF-3 Pro-Caspase-1 NOD2 and infected cells. Ten TLRs, 23 NLRs, and 3 RLRs Pro-Caspase-1cIAP-1 or -2 IPAF RIP2 IPAF ASC have been identi ed in humans to date, with Proteasome TLR9 Viral dsRNA P ASC Pro-Caspase-1 IKK ASC TAB1/2 Host Defense, and Inflammation only a subset of well-characterized NLR (NOD1, IRF3 IκB MKK P P Proteasome TLR7 Pro-Caspase-1 TAK1 Active Caspase-1 IRF3 IRF7 MyD88 Pro-Caspase-1 IKK ASC NOD2, NALP1, NALP3, IPAF) and RLR (RIG-1, P ASC CELL DEATH TLR8 Proteasome IκB MDA5) family members shown here. These PRRs IRF7 ASCAIM2 Pro-Caspase-1 IRF3 IRF7 ASCAIM2 Pro-Caspase-1 P TLR3 Pro-IL-1β, IL-18 Homo- Homo- are characterized by a variable number of li- IκB MyD88 IκB dimer dimer TRAF-3 P gand-sensing leucine-rich repeats (LRR) at their NFκB TRIF ASCAIM2 p38 TANK ASCAIM2 IκB JNK Mature IL-1β, IL-18 NFκB N-terminal (TLRs), or C-terminal (NLRs) ends, IRF3 Homodimer TAB1/2 and one or more protein-protein interaction TAK1 TRAF-6 IRF7 Homodimer P κ MKK INFLAMMATORY RESPONSE (TIR, CARD, PYR), or oligomerization (NACHT) Proteasome I B JNK RIP1 IKKε NFκB domain( s). Genetic variations that alter the IκB TBK1 functions of PRRs are associated with increased p38 IKK MKK susceptibility to infectious diseases (TLRs), as ERK P P IRF7 well as autoin ammatory (NLRs) and autoim- IκB JNK mune (TLRs and NLRs) disorders. NFκB IRF3 P IRF3 NFκBAP-1 IRF3 or IRF7 p38 AP-1 NFκB IRF3 or IRF7 Proinflammatory Cytokines: Proinflammatory Cytokines: Type I or III IFN & IFN-inducible genes IL-1β, TNF-α, IL-6, IL-8, IL-18 IRF3 Homodimer IL-1β, TNF-α, IL-6, IL-8, IL-18 This illustration represents general processes suggested in the scienti c Type I or III IFN & IFN-inducible genes literature and is not to be considered comprehensive nor de nitive. IRF7 Homodimer 2009, 2015 R&D Systems P_PatternRecog_

Global infobio-techne.com techsupportbio-techne.com orth Aerica urope iddle ast Africa LEARN MORE hina info.cnbio-techne.com rndsystems.com/PRR Rest of orld bio-techne.com/fi nd-us/distributors TEL +1 612 379 2956 bio-techne.com TLR Antibodies for Flow Cytometry, Immunocytochemistry, Immunohistochemistry, or Western Blot Detection of Plasma Membrane TLRs

Relative ell uber Relative Relative ell uber Relative

R R Detection of TLR1 on Human Monocytes by Flow Detection of TLR2 in Human Monocytes by Flow Detection of TLR4 in RAW264.7 Cells by Cytometry. Human peripheral blood monocytes Cytometry. Human peripheral blood monocytes Immunocytochemistry. TLR4 was detected in the were stained with an APC-conjugated Goat Anti- were stained with a PE-conjugated Mouse Anti- immersion-fixed RAW264.7 mouse / Human TLR1 Antigen Affinity-purified Polyclonal Human TLR2 Monoclonal Antibody (R&D Systems, cell line using a Rabbit Anti-Mouse Antibody (R&D Systems, Catalog # FAB1484A; filled Catalog # FAB2616P; filled histogram) or a PE- TLR4 Monoclonal Antibody (R&D Systems, Catalog histogram) or an APC-conjugated Isotype Control conjugated Isotype Control (R&D Systems, Catalog # MAB27591) at 1 µg/mL for 3 hours at room (R&D Systems, Catalog # IC108A; open histogram). # IC0041P; open histogram). temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (R&D Systems, Catalog # NL004; red) and counterstained with DAPI (blue). Specific staining was localized to cell surfaces and cytoplasm.

Relative ell uber Relative

R

Detection of TLR5 on Human Monocytes by Flow Detection of TLR5 in Human Intestine by Cytometry. Human peripheral blood monocytes Immunohistochemistry. TLR5 was detected in were stained with an Alexa Fluor® 488-conjugated immersion-fixed, paraffin-embedded sections of Mouse Anti-Human TLR5 Monoclonal Antibody human intestine using a Mouse Anti-Human TLR5 (R&D Systems, Catalog # FAB6704G; filled Monoclonal Antibody (R&D Systems, Catalog # histogram) or an Alexa Fluor 488-conjugated Mouse MAB6704) at 15 µg/mL overnight at 4 °C. Tissue

IgG1 Isotype Control (R&D Systems, Catalog # was stained using the Anti-Mouse HRP-DAB Cell & IC002G; open histogram). Tissue Staining Kit (R&D Systems, Catalog # CTS002; brown) and counterstained with hematoxylin (blue). Lower panels show a lack of labeling when primary antibodies are omitted and the tissue is stained with only secondary antibody followed by incubation with detection reagents.

For more information, please visit | rndsystems.com/TLR Detection of Endosomal TLRs ouse hymus NR kDa R

Relative ell uber Relative

Relative ell uber Relative

R R Detection of TLR3 in A549 Cells by Flow Cytometry. Detection of TLR7 in Ramos Human Burkitt’s Detection of Mouse and Rat TLR7 by Western Blot. The A549 human lung carcinoma cell line was Lymphoma Cells by Flow Cytometry. The Ramos Lysates from mouse thymus tissue or the NR8383 stained with an APC-conjugated Goat Anti-Human human Burkitt’s lymphoma cell line was stained rat alveolar macrophage cell line were separated by TLR3 Antigen Affinity-purified Polyclonal Antibody with a PE-conjugated Mouse Anti-Human TLR7 SDS-PAGE and immunoblotted using 1 µg/mL of the (R&D Systems, Catalog # IC1487A; filled histogram) Monoclonal Antibody (R&D Systems, Catalog # Rat Anti-Mouse/Rat TLR7 Monoclonal Antibody or an APC-conjugated Isotype Control (R&D Systems, IC5875P; filled histogram) or a PE-conjugated (R&D Systems, Catalog # MAB7156) followed by an

Catalog # IC108A; open histogram). To facilitate Mouse IgG2A Isotype Control (R&D Systems, Catalog HRP-conjugated Anti-Rat IgG Secondary Antibody. A intracellular staining, cells were fixed with Flow # IC003P; open histogram). To facilitate intracellular specific band for TLR7 was detected at Cytometry Fixation Buffer (R&D Systems, Catalog # staining, cells were fixed with Flow Cytometry approximately 130 to 140 kDa under reducing FC004) and permeabilized with Flow Cytometry Fixation Buffer (R&D Systems, Catalog # FC004) conditions (as indicated). Permeabilization/Wash Buffer I (R&D Systems, and permeabilized with Flow Cytometry Catalog # FC005). Permeabilization/Wash Buffer I (R&D Systems, Catalog # FC005). R

Detection of TLR9 in Mouse Splenocytes by Flow Cytometry. Mouse splenocytes were stained with an APC-conjugated Rat Anti-Mouse B220/CD45R Monoclonal Antibody (R&D Systems, Catalog # FAB1217A) and either a (A) Rabbit Anti-Mouse TLR9 Monoclonal Antibody (R&D Systems, Catalog # MAB7960) or (B) Normal Rabbit IgG Control (R&D Systems, Catalog # AB-105-C), followed by a PE-conjugated Anti-Rabbit IgG Secondary Antibody (R&D Systems, Catalog # F0110). Rabbit IgG

DR

For more information, please visit | rndsystems.com/TLR Toll-like Receptor Antibodies from R&D Systems R&D Systems® antibodies undergo rigorous lot-specific quality control testing for all of the applications listed on our datasheets to ensure superior performance, reliability, and consistency. For more information, please visit rndsystems.com/Antibodies

Unconjugated Antibodies Fluorochrome-conjugated Antibodies for Flow Cytometry Alexa Fluor® Molecule Species Clone Catalog # (Applications) APC Fluorescein PE PerCP 488 700 H Polyclonal AF1484 (FC, WB) ✓ ✓ TLR1 M Polyclonal AF1475 (FC, WB) ✓ M 285923 MAB1475 (WB) H 383936 MAB2616 (B/N, FC) ✓ ✓ ✓ ✓ ✓ H Polyclonal AF2616 (E, FC, WB) TLR2 M 203325 MAB1530 (FC) ✓ ✓ M Polyclonal AF1530 (WB) H 512505 MAB1487 (WB) H Polyclonal AF1487 (WB) ✓ TLR3 M 313129 MAB3005 (WB) ✓ ✓ M Polyclonal AF3005 (WB) H 285227 MAB1478 (WB) H 610029 MAB14782 (WB) H 610017 MAB14783 (IHC) H 610015 MAB6248 (FC) ✓ ✓ ✓ ✓ ✓ TLR4 AF1478 (B/N, FC, ICC/IF, H Polyclonal IHC, WB) M 267518 MAB2759 (FC, ICC/IF) ✓ ✓ M 1203B MAB27591 (FC, ICC/IF) TLR5 H 624915 MAB6704 (FC, IHC) ✓ M 418601 MAB1533 (FC) ✓ ✓ TLR6 M Polyclonal AF1533 (WB) H 533707 MAB5875 (FC) ✓ ✓ ✓ TLR7 M/R 726606 MAB7156 (WB) TLR8 H 935166 MAB8999 (FC) H 229106 MAB3658 (FC) TLR9 H Polyclonal AF3658 (FC, IHC) ✓ M 1138D MAB7960 (FC, ICC/IF) TLR10 H 670719 MAB6619 (WB) TLR11 M 786404 MAB7640 (FC) ✓ ✓ TLR12 M 1229C MAB8086 (FC, WB) ✓ Application Key: B/N Block/Neutralize E ELISA FC Flow Cytometry ICC/IF Immunocytochemistry/Immunofluorescence IHC Immunohistochemistry WB Western Blot

For more information, please visit | rndsystems.com/TLR Additional Select Toll-like Receptor Antibodies from Novus Biologicals Novus Biologicals offers a wide selection of TLR antibodies including some of the most highly referenced clones on the market. Most of these antibodies are conjugated to multiple different fluorochromes including a series of Alexa Fluor® and DyLight® dyes to provide a full range of options for multicolor experiments. For more information, please visit novusbio.com

Unconjugated Antibodies Fluorochrome-conjugated Antibodies for Flow Cytometry Alexa Fluor® Molecule Species Clone Catalog # (Applications) APC Fluorescein PE PerCP 405 488 647 700 NB100-56563 TLR1 H/M/R Polyclonal (FC, IHC, WB) NB100-56722 (B/N, FA, H TL2.1 ✓ ✓ ✓ ✓ ✓ ✓ ✓ FC, ICC/IF, IHC, IP) TLR2 H/M T2.5 NBP1-42362 (FC, IHC, IP) ✓ M 11G5 NBP2-27165 (FC, WB)* ✓ ✓ ✓ ✓ ✓ ✓ ✓ NBP2-24875 (FC, ICC/IF, H/M 40C1285.6 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ TLR3 IHC, IP, WB)* H/M TLR3.7 ✓ ✓ NB100-56723 (B/N, FA, H HTA125 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ FC, ICC/IF, IP, IV)* TLR4 NB100-56566 (FC, ICC/IF, H/M 76B357.1 ✓ ✓ ✓ ✓ ✓ IHC, ChIP, WB)* M MTS510 NB100-56560 (FC, IP)* ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ H/M 85B152.5 NBP1-97728 (FC, WB)* ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ TLR5 NBP2-24787 (FC, IHC, H/M 19D759.2 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ WB)* H 86B1153.2 NB100-56536 (FC, IHC)* ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ H hPer6 NBP1-43142 (FC, WB)* ✓ ✓ ✓ ✓ ✓ ✓ ✓ TLR6 NBP1-51493 (FC, ICC/IF, H TLR6.127 IHC, IP) NBP2-27332 (FC, ICC/ H/M 4G6 ✓ ✓ ✓ ✓ ✓ ✓ ✓ IF, WB)* TLR7 NBP2-24906 (FC, ICC/IF, H/M Polyclonal IHC, WB) NBP2-24917 (FC, IHC, H/M 44C143 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ SW, WB)* TLR8 NBP1-77203 (E, ICC/ H/M Polyclonal IF, WB) NBP1-43140 (FC, IHC, H eB72-1665 ✓ ✓ ✓ ✓ ✓ ✓ ✓ IP, WB)* NBP2-24729 (E, FA, FC, TLR9 H/M/R 26C593.2 ICC/IF, IHC, IP, IV, SW, ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ WB)* M M9.D6 NBP1-43141 (FC, WB)* ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ TLR10 H 3C10C5 NBP1-70343 (FC)* ✓ ✓ ✓ ✓ ✓ ✓ ✓ TLR13 M/R Polyclonal NBP2-24539 (FC, WB) Application Key: B/N Block/Neutralize ChIP Chromatin Immunoprecipitation E ELISA FA Functional Assay FC Flow Cytometry ICC/IF Immunocytochemistry/Immunofluorescence IHC Immunohistochemistry IP Immunoprecipitation IV In vitro WB Western Blot

* In addition to the fluorochrome-conjugated forms listed, these antibodies are also available in several DyLight®-conjugated forms. DyLight conjugates include DyLight 350, 405, 405LS, 488, 550, 650, 680, and 755. Please visit novusbio.com for more information.

For more information, please visit | novusbio.com Proteins & Antibodies for TLR Co-Receptors, Signaling Regulators, and Adaptor Proteins

TLR co-receptors such as CD14, CD36, and MD-2 have essential roles in ligand recognition, while intracellular, TIR domain-containing signaling adaptors such as MyD88, TRIF, TRAM, and TIRAP are required for activating signaling pathways downstream of TLRs. Additionally, molecules such as RP105/CD180, SARM1, PRAT4A, and PRAT4B are involved in regulating TLR signaling or TLR localization. R&D Systems and Novus Biologicals provide high quality antibodies to detect these proteins by multiple different methods.

P Antibody g - I- Secretion ean D . .

. . . . Protein . Antibody .

Side Scatter . . . . I- Secretion ean D Secretion I- I- Secretion ean D I- Secretion - . . D ng D P ng Recombinant Human CD14 Enhances LPS- Detection of CD14 in Human Peripheral Blood Recombinant Mouse LBP-Induced IL-8 Secretion Stimulated IL-8 Secretion by THP-1 Cells. The THP- Mononuclear Cells by Flow Cytometry. Human and Neutralization using an Anti-Mouse LBP 1 human acute monocytic leukemia cell line was peripheral blood mononuclear cells were stained Antibody. The THP-1 human acute monocytic treated with 15 ng/mL lipopolysaccharide (LPS) with an APC-conjugated Mouse Anti-Human CD14 leukemia cell line was treated with 5 ng/mL and the indicated concentrations of Recombinant Monoclonal Antibody (R&D Systems, Catalog # lipopolysaccharide and the indicated concentrations Human CD14 (R&D Systems, Catalog # 383-CD). FAB3832A). of Recombinant Mouse Lipopolysaccharide-Binding CXCL8/IL-8 secretion was measured using the Protein (LBP; R&D Systems, Catalog # 6635-LP). Human CXCL8/IL-8 Quantikine® ELISA Kit (R&D CXCL8/IL-8 secretion was measured using the ® Systems, Catalog # D8000C). The ED50 for this Human CXCL8/IL-8 Quantikine ELISA Kit effect is typically 0.75–4.5 ng/mL. (R&D Systems, Catalog # D8000C; orange line). The stimulatory effect induced by 50 ng/mL Recombinant Mouse LBP was neutralized by treating the cells with increasing concentrations of a Rat Anti-Mouse LBP Monoclonal Antibody (R&D Systems, Catalog # MAB6635; blue line). The

ND50 is typically 1−5 µg/mL.

A B

Relative ell uber Relative

yD

Detection of SARM1 in HEK293 Cells by Detection of MyD88 by Immunocytochemistry and Flow Cytometry. (A) MyD88 was detected in immersion- Immunocytochemistry. SARM1 was detected in the fixed RAW264.7 mouse monocyte/macrophage cell line using a Goat Anti-Mouse/Rat MyD88 Antigen immersion-fixed HEK293 human embryonic kidney Affinity-purified Polyclonal Antibody (R&D Systems, Catalog # AF3109) at 1.7 µg/mL for 3 hours at room cell line using a Sheep Anti-Human SARM1 Antigen temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Affinity-purified Polyclonal Antibody (R&D Systems, Antibody (R&D Systems, Catalog # NL001; red) and counterstained with DAPI (blue). Specific staining was Catalog # AF7037) at 1.7 µg/mL for 3 hours at localized to the cytoplasm. (B) MyD88 was detected in mouse splenocytes by staining with the Goat Anti- room temperature. Cells were stained using the Mouse/Rat MyD88 Antigen Affinity-purified Polyclonal Antibody (R&D Systems, Catalog # AF3109; filled NorthernLights™ 557-conjugated Anti-Sheep IgG histogram) followed by staining with an APC-conjugated Anti-Goat IgG Secondary Antibody (R&D Systems, Secondary Antibody (R&D Systems, Catalog # Catalog # F0108). To facilitate intracellular staining, cells were fixed with paraformaldehyde and NL010; red) and counterstained with DAPI (blue). permeabilized with saponin. Specific staining was localized to the cytoplasm. For more information, please visit | rndsystems.com/TLR Recombinant Proteins & Antibodies for TLR Co-Receptors, Signaling Regulators, & Adaptor Proteins from R&D Systems

Proteins Unconjugated Antibodies Fluorochrome-conjugated Antibodies for Flow Cytometry

Catalog # *Alexa Fluor® Molecule Species Catalog # Species Clone APC Fluorescein PE PerCP (Applications) 405/594/647/700/750

MAB3832 (B/N, Human 383-CD Human 134620 ✓ ✓ ✓ ✓ ✓ CD14 FC, WB) Mouse 982-CD Mouse 159010 MAB982 (FC, WB) ✓ ✓ ✓ Human 255606 MAB19551 (FC) ✓ ✓ ✓ Human 1955-CD Human 255619 MAB1955 (WB) CD36 MAB25191 (FC, Mouse 324205 ✓ Mouse 2519-CD IHC) Mouse 324216 MAB2519 (WB) Human 870-LP Human Polyclonal AF870 (WB) LBP Human 6445-LP Mouse 749405 MAB6635 (B/N) Mouse 6635-LP Mouse Polyclonal AF6635 (WB) AF925 (FC, IHC, Human Polyclonal Human 925-MD WB) MD-1 Human 153014 MAB925 (WB) Mouse 142004 MAB130 (WB) Mouse 130-MD Mouse Polyclonal AF130 (WB) Human 288307 MAB1787 (WB) MD-2 Human 1787-MD Human Polyclonal AF1787 (WB) Human 316628 MAB29281 (FC) Human 316603 MAB2928 (ICC/IF) AF2928 (FC, ICC/ Human Polyclonal MyD88 IF, SW, WB) Mouse 316902 MAB3109 (ICC/IF) AF3109 (FC, ICC/ Detection of TRAM/TICAM2 in C2C12 Cells by Mouse Polyclonal IF, SW, WB) Immunohistochemistry. TRAM/TICAM2 was detected in the immersion-fixed C2C12 mouse Human 7484-PR PRAT4A myoblast cell line using a Rat Anti-Mouse TRAM/ Mouse 4429-PR Mouse Polyclonal AF4429 (WB) TICAM2 Monoclonal Antibody (R&D Systems, Catalog # MAB43481) at 10 ug/mL for 3 hours at PRAT4B Mouse Polyclonal AF5015 (FC, WB) room temperature. Cells were stained using the RP105/ Mouse 1378-RP NorthernLights™ 557-conjugated Anti-Rat IgG CD180 Secondary Antibody (R&D Systems, Catalog # Human 754711 MAB7037 (ICC/IF) NL013; red) and counterstained with DAPI (blue). Specific staining was localized to the cytoplasm. SARM1 AF7037 (ICC/IF, Human Polyclonal IP, WB) Human/ AF4348 (FC, ICC/ Mouse/ Polyclonal IF, WB) Rat TRAM/TI- CAM2 Mouse 757706 MAB4348 (WB) MAB43481 (ICC/ Mouse 757712 IF) Human 567212 MAB6216 (WB) TRIF AF6216 (ICC/ Human Polyclonal IF, WB) Application Key: B/N Block/Neutralize FC Flow Cytometry ICC/IF Immunocytochemistry/ Immunofluorescence IHC Immunohistochemistry IP Immunoprecipitation SW Simple Western WB Western Blot

*Indicates one or more of the following conjugates is available.

For more information, please visit | rndsystems.com/TLR Additional Select Antibodies for TLR Co-Receptors, Signaling Regulators, & Adaptor Proteins from Novus Biologicals

Unconjugated Antibodies Fluorochrome-conjugated Antibodies for Flow Cytometry Alexa Fluor® Molecule Species Clone Catalog # (Applications) APC Fluorescein PE PerCP 405 488 647 700 Human HCD14 NB100-78083 (FC)* ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ CD14 Human M5E2 NB100-77758 (FC, ICC/IF, IHC)* ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Human SM0 NB100-65522 (FC, IHC)* ✓ ✓ ✓ ✓ ✓ ✓ ✓ CD36 Mouse/Rat D-2712 NB110-59724 (IHC, IP, WB)* ✓ ✓ ✓ ✓ ✓ LBP Human Polyclonal NBP1-88371 (IHC) Human/ Polyclonal NB100-56700 (IHC, WB) MD-1 Mouse Mouse MD-14 NB100-77635 (FC) Human/ Polyclonal NB100-56655 (IHC, WB) Mouse/Rat Human/ MD-2 Polyclonal NBP1-77201 (E, ICC/IF, IHC, WB) Mouse/Rat Human/ 9F1B1 NBP1-75512 (E, ICC/IF, IHC, WB) Mouse Human/ MyD88 4D6 NBP2-27369 (FC, ICC/IF, WB)* ✓ ✓ ✓ ✓ ✓ ✓ ✓ Mouse Human/ Polyclonal NBP1-91153 (ICC/IF, IHC, WB) Mouse/Rat PRAT4A Human/ Polyclonal NBP2-23847 (IHC) Mouse/Rat PRAT4B Human Polyclonal NBP1-81085 (ICC/IF, IHC, WB) RP105/CD180 Human MHR73-11 NBP1-49025 (FC, IHC, IP)* ✓ ✓ ✓ ✓ ✓ ✓ ✓ Human/ SARM1 Polyclonal NBP1-77200 (E, ICC/IF, IHC, WB) Mouse Human 20D1055.1 NB100-56730 (WB) TIRAP/MAL Human/ Polyclonal NBP2-47605 (ICC/IF, IHC) Mouse Human Polyclonal NBP1-89566 (IHC) TRIF Human/ Polyclonal NB120-13810 (ICC/IF, SW, WB) Mouse/Rat Human/ UNC93B Polyclonal NBP2-24743 (WB) Mouse/Rat Application Key: E ELISA FC Flow Cytometry ICC/IF Immunocytochemistry/Immunofluorescence IHC Immunohistochemistry IP Immunoprecipitation SW Simple Western WB Western Blot

*In addition to the fluorochrome-conjugated forms listed, these antibodies are also available in several DyLight®-conjugated forms. DyLight conjugates include DyLight 350, 405, 405LS, 488, 550, 650, 680, and 755. Please visit novusbio.com for more information.

For more information, please visit | novusbio.com Antibodies for Detecting TLR Signaling Molecules

Following ligand recognition by TLRs, downstream cascades result in the activation of several kinases including IKK, MKK, p38 MAPK, JNK, and TBK1. These kinases activate transcription factors such as AP-1, NFkB, IRF3, and IRF7, which promote the expression of pro-inflammatory cytokines, chemokines, and type I and type III interferons. R&D Systems and Novus Biologicals offer antibodies to detect the kinases and transcription factors involved in TLR signaling, while Tocris offers inhibitors and activators of these molecules. Together, these reagents can be used to fully characterize TLR signaling pathways.

A B

kDa rhIRA rhIRA rhIRA A IRA Relative ell uber Relative

RelAκ p

Intracellular Staining of RelA/NFkB p65 in HeLa Detection of IRAK4 by Western Blot and Immunocytochemistry. (A) Recombinant Human IRAK1, IRAK2, Cells by Flow Cytometry. HeLa human cervical IRAK4 (2 ng/lane), and lysates from the A431 human epithelial carcinoma cell line or the K562 human epithelial carcinoma cells were stained with an chronic myelogenous leukemia cell line were separated by SDS-PAGE and immunoblotted using 1 µg/mL of Alexa Fluor® 488-conjugated Mouse Anti-Human/ the Goat Anti-Human IRAK4 Antigen Affinity-purified Polyclonal Antibody (R&D Systems, Catalog # AF3919) Mouse RelA/NFkB p65 Monoclonal Antibody followed by an HRP-conjugated Anti-Goat IgG Secondary Antibody (R&D Systems, Catalog # HAF109). A (R&D Systems, Catalog # IC5078G; filled histogram) specific band for IRAK4 was detected at approximately 55 kDa under reducing conditions (as indicated).(B) or an Alexa Fluor 488-conjugated Mouse IgG2B IRAK4 was detected in the immersion-fixed THP-1 human acute monocytic leukemia cell line using the Goat Isotype Control (R&D Systems, Catalog # IC0041G; Anti-Human IRAK4 Antigen Affinity-purified Polyclonal Antibody (R&D Systems, Catalog # AF3919) at open histogram). 15 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (R&D Systems, Catalog # NL001; red) and counterstained with DAPI (blue). Specific staining was localized to the cytoplasm.

Sample-Size Antibodies Now Available Choose From Over 10,000 Antibodies

Learn more and find products | rndsystems.com/samplesize R&D Systems® Antibodies for TLR Signaling Molecules Tocris Small Molecule Activators/Inhibitors Unconjugated Antibodies Available Molecule Species Catalog # (Applications) Human MAB4299 (SW, WB) X IkB-a Human/Mouse AF4299 (WB) Phospho-IkB-a (S32/S36) Human AF4809 (WB) Human/Rat MAB3425 (WB) IkB-b Human/Mouse AF5225 (WB) Human MAB4300 (WB) IkB-e Human AF4300 (IHC) Mouse AF4637 (WB) IKK-a Human/Mouse/Rat AF3768 (ICC/IF, WB) X Phospho-IKK-a (S176/S180) Human MAB3768 (WB) Human AF4535 (WB) X IKK-b Mouse MAB7155 (WB) Human/Mouse/Rat AF2684 (ICC/IF, SW, WB) X IKK-g Human/Mouse/Rat AF4365 (WB) Human AF3199 (ICC/IF, WB) IKK-e Human/Mouse/Rat MAB3199 (ICC/IF, WB) IRAK1 Human AF4048 (WB) X IRAK2 Human MAB6690 (WB) IRAK3 Human AF6264 (WB) IRAK4 Human AF3919 (ICC/IF, WB) X Human/Mouse/Rat AF1387 (IHC, WB) X JNK Pan Specific Human/Mouse/Rat MAB1387 (WB) Human/Mouse/Rat AF1205 (IHC, SW, WB) Phospho-JNK (T183/Y185) Human/Mouse/Rat MAB1205 (ICC/IF, SW, WB) Human/Mouse/Rat MAB17761 (ICC/IF, WB) X JNK1 Human/Mouse/Rat MAB1776 (WB) JNK1/2 Human/Mouse/Rat MAB2076 (ICC/IF, WB) Human/Mouse/Rat MAB1846 (ICC/IF, WB) X JNK2 Human/Mouse/Rat AF1846 (WB) MKK3 Human/Mouse/Rat MAB2515 (ICC/IF, WB) MKK3/MKK6 Human/Mouse/Rat MAB2514 (WB) MKK4 Human MAB3390 (ICC/IF) Phospho-MKK4 (S257/T261) Human/Mouse/Rat AF2990 (ICC/IF, WB) Human/Mouse/Rat MAB1604 (ICC/IF, WB) MKK6 Human/Mouse/Rat AF16041 (WB) Human/Mouse/Rat AF1604 (WB) Human AF3579 (IHC, WB) MKK7 Human MAB3579 (IHC) Human/Mouse/Rat AF8691 (IHC, SW, WB) X p38a Human/Mouse/Rat MAB869 (WB) Human MAB8691 (WB) Phospho-p38a (T180/Y182) Human MAB8692 (ICC/IF) Human/Mouse/Rat AF1347 (IHC, SW, WB) X p38g Human/Mouse/Rat MAB1347 (SW, WB) Human/Mouse/Rat AF1644 (WB) Human AF1519 (IHC, WB) X p38d Human MAB1519 (WB) RIP1/RIPK1 Human/Mouse/Rat MAB3585 (SW, WB) X TAB1 Human/Mouse AF3578 (ICC/IF, WB) TAK1 Human MAB5307 (WB) TANK Human/Mouse AF4755 (WB) Human AF3278 (WB) TRAF-3 Isoform 2 Human/Mouse/Rat MAB3278 (WB) TRAF-6 Human AF3284 (WB) Application Key: ICC/IF Immunocytochemistry/Immunofluorescence IHC Immunohistochemistry SW Simple Western WB Western Blot

For more information, please visit | rndsystems.com/TLR R&D Systems® Antibodies for TLR-related Transcription Factors

Tocris Small Molecule Activators/ Unconjugated Antibodies Fluorochrome-conjugated Antibodies Inhibitors Available Molecule Species Catalog # (Applications) APC Alexa Fluor® 488 PE c-Fos Human AF7254 (WB) Human AF2214 (IHC, WB) FosB/GOS3 Human/Mouse MAB2214 (WB) Human AF4935 (IHC, WB) FRA-1 Human MAB4935 (WB) Human AF4019 (FC, ICC/IF, WB) IRF3 Human MAB4019 (FC, WB) ✓ ✓ ✓ Mouse AF4454 (WB) Human MAB2670 (ICC/IF, WB) X c-Jun Human AF2670 (WB) Phospho-c-Jun (S63) Human MAB8930 (ICC/IF, SW, WB) Human MAB4456 (WB) JunB Human AF4456 (WB) Human/Mouse MAB5526 (WB) JunD Human/Mouse AF5526 (WB) Human MAB2697 (WB) X NFkB1 Human/Mouse AF2697 (ChIP, WB) MAB28881 (ChIP, ICC/ NFkB2 Human X IF, WB) Human MAB4606 (ICC/IF, WB) AF2699 (ChIP, ICC/IF, SW, c-Rel Human/Mouse WB) Mouse MAB2699 (WB) Human MAB50781 (FC) RelA/NFkB p65 Human/Mouse MAB5078 (FC, ICC/IF, WB) ✓ ✓ ✓ Human/Mouse AF5078 (ChIP, SW, WB) Phospho-RelA/NFkB p65 Human MAB7624 (WB) (S529)

Phospho-RelA/NFkB p65 Human MAB7226 (ICC/IF, WB) (S536) Human MAB72261 (ICC/IF, SW, WB) RelB Human MAB2698 (ICC/IF, IHC, WB) Application Key: FC Flow Cytometry ICC/IF Immunocytochemistry/Immunofluorescence IHC Immunohistochemistry ChIP Chromatin Immunoprecipitation SW Simple Western WB Western Blot

For more information, please visit | rndsystems.com/TLR Additional Select Antibodies for TLR Signaling Molecules from Novus Biologicals

Unconjugated Antibodies Fluorochrome-conjugated Antibodies Molecule Species Catalog # (Applications) APC PE PerCP Alexa Fluor® 405 488 647 700 NB100-56507 (FC, ICC/ IkB-a Human/Mouse/Rat ✓ ✓ ✓ ✓ ✓ ✓ ✓ IF, IHC, IP, SW, WB)* NB100-56704 (FC, ICC/ IKK-a Human/Mouse ✓ ✓ ✓ ✓ ✓ ✓ ✓ IF, IHC, IP, SW, WB)* NB100-56509 (FC, ICC/ IKK-b Human/Mouse ✓ ✓ ✓ ✓ ✓ ✓ ✓ IF, IHC, IP, SW, WB)* IRAK4 Human/Mouse/Rat NB500-597 (FC, IP, WB) NBP1-77077 (E, ICC/IF, RIP1 Human/Mouse/Rat IHC, WB) NB100-56176 (IHC, TRAF-3 Human/Mouse/Rat IP, WB) Human/Mouse NB100-56436 (FC, WB) TRAF-6 NB100-56179 (IHC, Human/Mouse/Rat IP, WB) Application Key: E ELISA FC Flow Cytometry ICC/IF Immunocytochemistry/Immunofluorescence IHC Immunohistochemistry IP Immunoprecipitation SW Simple Western WB Western Blot

*In addition to the fluorochrome-conjugated forms listed, these antibodies are also available in several DyLight®-conjugated forms. DyLight conjugates include DyLight 350, 405, 405LS, 488, 550, 650, 680, and 755. Please visit novusbio.com for more information.

Additional Select Antibodies for TLR-related Transcription Factors from Novus Biologicals

Unconjugated Antibodies Fluorochrome-conjugated Antibodies Molecule Species Catalog # (Applications) Alexa Fluor® 405 488 647 700 c-Fos Human/Mouse/Rat NBP1-89065 (ICC/IF, IHC, WB) IRF7 Human/Mouse/Rat NBP1-77263 (E, ICC/IF, IHC, WB) c-Jun Human/Mouse/Rat NB110-55569 (FC, ICC/IF, IHC, IP, WB) NFkB1 Human/Mouse NBP1-77395 (ICC/IF, IHC, SW, WB) ✓ ✓ ✓ ✓ NFkB2 Human/Mouse/Rat NBP1-87760 (ICC/IF, IHC, SW, WB) Application Key: E ELISA FC Flow Cytometry ICC/IF Immunocytochemistry/Immunofluorescence IHC Immunohistochemistry IP Immunoprecipitation SW Simple Western WB Western Blot

For more information, please visit | novusbio.com ELISA Kits for Select Cytokines Induced by TLRs

For detecting cytokines and chemokines that are induced by TLR signaling, R&D Systems offers complete, ready-to-run Quantikine® ELISA Kits and the more flexible DuoSet® ELISA Development Systems.

Quantikine® ELISA Kit Features

• Complete, ready-to-use kits • Detailed protocol booklets

• Exhaustively tested for superior quality and reproducibility • Colorimetric detection Assay Principle Step 1 Step 2 Step 3 nalyte TMB Sustrate RP-conugated Detection ntiody

RP RP

RP RP RP

ntiody-coated TMB Microplate RP RP Sustrate

Blue

Stop Capture ntiody ellow

A microplate pre-coated with capture antibody is provided. Samples or standards are added and any analyte present is bound by the immobilized antibody. Unbound materials are washed away (Step 1). A second HRP-labeled detection antibody is added and binds to the captured analyte. Unbound detection antibody is washed away (Step 2). Tetramethylbenzidine (TMB) substrate solution is added to the wells and a blue color develops in proportion to the amount of analyte present in the sample. Color development is stopped turning the color in the wells to yellow. The absorbance of the color at 450 nm is measured (Step 3).

DuoSet® ELISA Development System Features

When complete kits are not an option, DuoSet® ELISA Development Systems offer an economical alternative. DuoSet® Kits contain the essential components required to develop an immunoassay, but unlike Quantikine® ELISA Kits, they require the user to set up the assay by coating a microplate with the provided capture antibody. DuoSet® Kits also provide a biotinylated detection antibody and streptavidin-HRP, enabling chemiluminescent or colorimetric detection, a mass-calibrated standard, and detailed protocol. • Contains all of the essential components required to develop an immunoassay for a specific target

• Contains carefully selected and validated antibodies, reducing development time

• Provides sufficient reagents for five or fifteen 96-well plates

• Includes mass-calibrated recombinant standard, reducing assay variability

• Can be adapted for use across multiple platforms

Molecule Species Quantikine® ELISA Kits Catalog # Quantikine® High Sensitivity ELISA Kits Catalog # DuoSet® ELISA Development Systems Catalog # Human DIF50 DY285 IFN-g Mouse MIF00 DY485 Human DLB50 HSLB00C DY201 IL-1b/IL-1F2 Mouse MLB00C DY401 Human D6050 HS600B DY206 IL-6 Mouse M6000B DY406 IL-8/CXCL8 Human D8000C HS800 DY208 Human DTA00C HSTA00D DY210 TNF-a Mouse MTA00 DY410 For more information, please visit | rndsystems.com/ELISAs Multiplex Assays from R&D Systems

R&D Systems also offers Proteome Profiler™ Antibody Arrays and Luminex® Bead-Based Assays for simultaneously profiling the levels of multiple analytes in a single sample. Multiplexing allows researchers to maximize data collection from small sample volumes, minimize experimental variability, and optimize productivity.

Proteome Profiler™ Antibody Arrays Proteome Profiler Antibody Arrays are available for determining the relative levels of phosphorylated MAPKs, NFkB-related analytes, chemokines, or cytokines in a single sample.

ntreated P- ntreated vs I-γ PS reated /RANS

P- P- IFN-γ PS

I-

I-γ PS reated /I-A /RANS /IP-

/ / / I- I- RANS IP- I-A

I- I- Simultaneous Detection of Multiple Analytes in IFN-g-, LPS-treated THP-1 Cell Lysates using the Proteome Profiler™ Human Cytokine Array. The THP-1 human acute monocytic leukemia cell line was untreated or treated with 1 µg/mL Recombinant Human IFN-g (R&D Systems, Catalog # 285-IF) for 8 hours and 1 µg/mL lipopolysaccharide (LPS) for 16 hours. Cytokine expression in 200 µg of cell lysate was analyzed using the Proteome Profiler Human Cytokine Array (R&D Systems, Catalog # ARY005B).

Select Proteome Profiler™ Antibody Arrays from R&D Systems

Kit Catalog # Proteome Profiler Human Phospho-MAPK Array Kit ARY002B Proteome Profiler Human NFkB Pathway Array Kit ARY029 Proteome Profiler Human Cytokine Array Kit ARY005B Proteome Profiler Human XL Cytokine Array Kit ARY022 Proteome Profiler Human Chemokine Array Kit ARY017 Proteome Profiler Mouse Cytokine Array Kit, Panel A ARY006 Proteome Profiler Mouse XL Cytokine Array Kit ARY028 Proteome Profiler Mouse Chemokine Array Kit ARY020

Please visit rndsystems.com/ProteomeProfilerfor a list of analytes detected by each array and the qualified sample types.

For more information, please visit | rndsystems.com/proteomeprofiler Luminex® Assays R&D Systems offers two versions of our Luminex® bead-based assays. Our standard Luminex Assays offer the largest, customizable menu of analytes for bead-based multianalyte profiling Analyte using cell culture supernatnants, serum, or plasma samples. These assays allow up to 100 analytes to be simultaneously profiled using polystyrene microparticles or 50 analytes using magnetic particles. Please visit rndsystems.com/LuminexAssay to see our industry-leading selection of analyte combinations. Streptavidin-P iotin Detection Antibody Luminex High Performance Assays offer defined analyte panels for bead-based multianalyte arget Analyte apture Antibody profiling. These assays rely on panel-optimized diluents that provide maximum performance for a smaller group of analytes than our standard Luminex Assays. Each assay is fully umine ead validated for all sample types indicated for a given panel. In-house testing demonstrates that analyte concentrations determined using our Luminex High Performance Assays correlate closely with those obtained using our single analyte Quantikine® ELISA Kits. Please visit rndsystems.com/LuminexPerformance to view a list of available kits.

Custom Services from Bio-techne

When your work demands unique reagents or scientific support, turn to the decades of product development legacy behind Bio-techne’s trusted brands. Together with a dedicated project manager, our expert scientists, quality assurance team, and world-class technical support, we will deliver custom solutions tailored to bring you success faster and more economically.

Benefits of Custom Services from Bio-Techne What You Can Expect

• Scientific expertise • Identify the need

• Consistency • Consult with our experts

• Supply • Refine the project specifics, milestones, and deliverables • Large-scale production • Review a statement of work • Regulatory support • Receive regular project updates • Quality results • Accept delivery of custom product or service • Timeliness

• ISO-certified Quality Management System and FDA registered

• Long-term cost savings

• Confidentiality

• Dedicated project managers

Custom Services

• Protein Services

• Assay Services

• Antibody Services

• Recombinant Antibody Conversion

• Sister Clone Availability

• Luminex® Custom Services

• Biomarker Testing Service

• Chemistry Services

• Custom Compound Libraries from Tocris

• Bioactivity Testing Services

• /Proteasome Custom Services Learn more | rndsystems.com/services ocri- RnDSy- ovus-

Global [email protected] bio-techne.com/find-us/distributors TEL +1 612 379 2956 North America TEL 800 343 7475 Europe | Middle East | Africa TEL +44 (0)1235 529449 China [email protected] TEL +86 (21) 52380373 bio-techne.com

For research use or manufacturing purposes only. Trademarks and registered trademarks are the property of their respective owners. BR_TLR AAI2016_2139

Top View