R&D Systems Tools for Cell Biology Research™

The TNF Superfamily The TNF The Superfamily

TNF Homology Domain Ribbon Structure The TNF Superfamily Ligands Receptors The tumor necrosis factor (TNF) superfamily in humans currently consists of 19 ligands and 29 receptors, with three additional TNF family receptors 4-1BB Ligand having been identifi ed in mice. Most TNF ligands are type II trans- membrane proteins with extracellular domains that can be cleaved by 4-1BB specifi c metalloproteinases to generate soluble cytokines. Cleaved and APRIL BCMA non-cleaved ligands are active as noncovalent homotrimers except for Lymphotoxin , which forms heterotrimers with TNF-/Lymphotoxin  BAFF TACI (LT) and BAFF, which forms heterotrimers with APRIL. TNF family ligands are characterized by a stalk of varying length connecting the BAFF R transmembrane domain to the core region, which contains the hallmark CD27 structure of TNF family ligands, the TNF homology domain (THD). The CD27 Ligand CD30 THD is an anti-parallel -pleated sheet sandwich with a “jelly-roll” topology. Conserved residues within the  strands provide specifi c inter- CD40 subunit contacts, which stabilize the trimeric structure. Sequences in the EDAR loops connecting adjacent  strands are family member-specifi c and are CD30 Ligand important for conferring receptor specifi city. Receptors for TNF family XEDAR ligands are oligomeric, type I or type III transmembrane proteins that contain multiple extracellular cysteine-rich domains (CRDs). Several of CD40 Ligand Fas these receptors contain intracellular death domains (DDs) that recruit EDA-A1 caspase-interacting proteins to initiate apoptosis upon ligand binding. DcR3 Other TNF superfamily receptors that lack DDs bind TNF receptor- EDA-A2 GITR associated factors (TRAFs) and activate multiple intracellular signaling Fas Ligand pathways that can lead to proliferation or diff erentiation. These receptors HVEM can also initiate apoptosis, but they do so via indirect mechanisms. GITR Ligand TNF superfamily ligands and receptors are important for numerous DR3 LIGHT processes that regulate immune cell functions including apoptosis, B cell homeostasis and activation, natural killer cell activation, T cell co- TL1A/VEGI LTER stimulation, and other cell type-specifi c responses such as hair follicle LTD1E2 development and osteoclast development. TNF superfamily members TWEAK R also play a signifi cant role in regulating the pathogenesis of human LTD E 2 1 TWEAK diseases including cancer, osteoporosis, osteoarthritis, chronic OX40 infl ammation, and autoimmune diseases.1,2 Nearly all TNF superfamily OX40 Ligand ligand-receptor pathways are being investigated as targets for the TNF RI development of agonist or antagonist therapeutics.2,3 TNF-D TNF RII R&D Systems off ers a wide selection of high quality reagents to TNF-E/LTD facilitate the study of TNF superfamily members including recombinant proteins, ELISA kits, and antibodies for blocking/neutralization, TRAIL R1 immunohistochemistry, immunoprecipitation, Western blotting, TRAIL TRAIL R2 and fl ow cytometry. For a complete, up-to-date listing and more information on these products, please visit our website at TRANCE/RANK Ligand TRAIL R3 www.RnDSystems.com/TNFSF. References 1. Aggarwal, B.B. et al. (2012) Blood 119:651. TRAIL R4 2. Tansey, M.G. & D.E. Szymkowski (2009) Drug Discov. Today 14:1082. Neurotrophins OPG 3. Hymowitz, S.G. & A. Ashkenazi (2005) Nat. Chem. Biol. 1:353. APP ? RANK ? LIGAND/RECEPTOR DOMAIN KEY SELECT FUNCTIONS NGF R TNF homology domain (THD) B cell homeostasis & activation Cysteine-rich domain (CRD) Natural killer cell activation DR6 Death domain (DD) T cell co-stimulation RELT Pro-apoptotic TROY Interactions with decoy receptors Diagrams include human family members only. Additional mouse TNF superfamily members include DcTRAIL R1, Other cell type-specific responses DcTRAIL R2, and TNFRH3.

www.RnDSystems.com TNF Superfamily Members Mediate B Cell Homeostasis and Activation Several TNF superfamily members play key roles in early B cell maturation, homeostasis, diff erentiation, and activation. While BAFF R is required for normal B cell maturation, interactions between BAFF- Ligands Receptors BAFF R, BAFF-TACI, APRIL-BCMA, and CD40 Ligand-CD40 promote B cell diff erentiation and 4-1BB Ligand homeostasis. The interaction between CD40 Ligand and CD40 is necessary for germinal center B cell 4-1BB diff erentiation, and CD27 and CD30 are memory B cell markers. Interactions between CD27 Ligand, BCMA APRIL CD30 Ligand, or CD40 Ligand and their respective receptors are also involved in B cell co-stimulation TACI by T cells and natural killer cells. Current research suggests roles for BAFF, APRIL, BAFF R, TACI, and BAFF R BCMA in immune defi ciency, autoimmune diseases, and lymphoid cell cancers.1-3 Mutations of the BAFF TACI and BAFF R genes are associated with common variable immunodefi ciency (CVID).1 Alternatively, BAFF overexpression is often observed in patients with autoimmune diseases such as lupus, CD27 Ligand CD27 rheumatoid arthritis, and multiple sclerosis, where it is thought that high levels of BAFF may lead to 2 the inappropriate survival of low-affi nity, self-reactive B cells. In addition, BAFF, APRIL, and their CD30 Ligand CD30 receptors are overexpressed in lymphoid cancers and their role in multiple myeloma is being 3 CD40 investigated. CD40 Ligand References 1. Eibel, H. et al. (2010) Curr. Opin. Allergy Clin. Immunol. 10:526. 3. Quinn, J. et al. (2011) Blood 117:890. 2. Mackay, F. & P. Schneider (2009) Nat. Rev. Immunol. 9:491.

Differentiation Differentiation TACI BAFF R Survival BCMA TACI APRIL TACI CD27 BCMA CD30 Memory B Cell Differentiation Plasma Cell TACI Plasma Cell BAFF R CD40 Ligand BCMA Proliferation CD40 TACI BAFF R Survival Germinal Center B Cell B Cell Development and Activation Requires TNF Superfamily Marginal Zone Members. Immature B cells (blue) express BAFF R prior to leaving the B Cell bone marrow. Mature B cells (purple) in the marginal zone and follicles Proliferation express BAFF R and TACI. CD27, CD30, and CD40 expressed on mature, Activation TACI BAFF R Differentiation naïve follicular B cells can mediate the thymus-dependent co-stimulatory TACI signal provided by T helper cells (yellow) that is necessary to activate the Differentiation BAFF R BAFF R CD40 Ligand B cell. Activated B cells maintain expression of TACI, BAFF R, and CD40. CD40 CD40 Ligand promotes activated B cell proliferation and diff erentiation as MHC II TCR they enter the germinal center. Following diff erentiation, plasma cells ex- CD27 CD27 Ligand press TACI and BCMA, which promote plasma cell survival. Similarly, TACI, Differentiation CD40 CD40 Ligand Survival CD30 BCMA, BAFF R, CD30, and CD27 are expressed by memory B cells and act to CD30 Ligand Activated B Cell B Cell Precursor Transitional B Cell Naïve B Cell maintain memory B cell homeostasis. Terminal processes are boxed, inter- T Helper Cell mediary processes are shown unboxed.

CD40/TNFRSF5 Antibody (ng/mL) 100 101 102 103 150 80 3500 3500

Cell Proliferation (Mean CPM) 70 120 3000 3000 60 50 2500 2500 90

2000 2000 40 60 30 1500 1500 Relative Cell Number Cell Relative Relative Cell Number Cell Relative 20

Cell Proliferation (Mean CPM) Proliferation Cell 30 1000 1000 10

500 500 0 0 10-1 100 101 102 100 101 102 103 104 100 101 102 103 104 CD40 Ligand/TNFSF5 (μg/mL) BAFF/TNFSF13B 4-1BB Ligand/TNFSF9 CD40 Ligand-induced Proliferation and Neutralization by an Anti-Human CD40 Detection of BAFF by Flow Cytometry. Mouse splenocytes were stained with a Detection of 4-1BB Ligand by Flow Cytometry. The A20 mouse B cell lymphoma cell Antibody. Proliferation of human B cell enriched peripheral blood mononuclear cells PE-conjugated Rat Anti-Mouse BAFF/TNFSF13B Monoclonal Antibody (Catalog line was stained with a PE-conjugated Rat Anti-Mouse 4-1BB Ligand/TNFSF9 was assessed following treatment with increasing concentrations of Recombinant # IC1357P; fi lled histogram) or a PE-conjugated Rat IgG2A Isotype Control (Catalog Monoclonal Antibody (Catalog # FAB1246P; fi lled histogram) or a PE-conjugated Rat

Human CD40 Ligand/TNFSF5 (Catalog # 6245-CL; orange line) in the presence of # IC006P; open histogram). IgG2B Isotype Control (Catalog # IC013P; open histogram). 20 ng/mL Recombinant Human IL-4 (Catalog # 204-IL). The stimulatory eff ect induced by 10 μg/mL Recombinant Human CD40 Ligand/TNFSF5 was neutralized by treating the cells with increasing concentrations of a Mouse Anti-Human CD40/TNFRSF5 Monoclonal Antibody (Catalog # MAB6322; green line).

For additional information, please visit our website at www.RnDSystems.com/TNFSFBCells TNF Superfamily Members Regulate Natural Killer Cells Upon activation, natural killer (NK) cells secrete cytotoxic and infl ammatory cytokines that cause Ligands Receptors apoptosis or lysis of targeted cells. While some pathogens can directly activate NK cells, NK cells are 4-1BB Ligand indirectly activated in response to most pathogens by accessory cells. Several TNF superfamily 4-1BB CD27 Ligand members have been implicated in mediating NK cell activation and regulation by accessory cells. CD27 These members include 4-1BB, CD27, CD30, CD40, and GITR and their respective ligands, as well as CD30 Ligand CD30 HVEM via LIGHT and TNF RII via TNF-or TNF-. NK cells also express Fas Ligand and TNF- on their cell surface that, when bound to target cell-expressed Fas or TNF RI, respectively, initiate caspase-mediated CD40 Ligand CD40 apoptosis of the target cell. TNF superfamily-mediated apoptosis and the ability of activated NK cells GITR Ligand GITR to initiate perforin/granzyme-mediated death of tumor cells prevents the formation of both solid 1 LIGHT tumors and lymphomas. The anti-cancer activity of 4-1BB-activated NK cells is currently being HVEM investigated as an adjuvant therapy in conjunction with traditional cancer treatments.2 TNF-D References TNF RII 1. Cullen, S.P. et al. (2010) Cell Death Diff er. 17:616. TNF-E/LTD 2. Kohrt, H.E. et al. (2012) J. Clin. Invest. 122:1066.

Accessory Cell Target Cell Apoptosis

Fas TNF RI 4-1BB CD27 CD30 CD40 GITR TNF-D Ligand Ligand Ligand Ligand Ligand LIGHT or TNF-E

Perforin/Granzymes

Fas Ligand TNF-D 4-1BB CD27 CD30 CD40 GITR HVEM TNF RII

Activation

Natural Killer Cell Activation and Target Cell Killing is Regulated by TNF Superfamily Members. Accessory cells (ACs) can express 4-1BB Ligand, CD27 Ligand, CD30 Ligand, CD40 Ligand, GITR Ligand, LIGHT, TNF-, or TNF- on their plasma membranes. TNF superfamily ligands on the ACs bind to their respective receptors on natural killer (NK) cells and activate these cells. In addition to facilitating perforin/granzyme- Natural Killer Cell mediated target cell death, activated NK cells express Fas Ligand and TNF- on their surfaces that can initiate caspase-mediated apoptosis upon contact with target cells expressing Fas or TNF RI.

40 2.3

30 1.8

20 1.3

Relative Cell Number Cell Relative 10 0.8 IL-8 Secretion (Mean O.D.) Secretion IL-8

0 0.3 0 1 2 3 4 10 10 10 10 10 10-2 10-1 100 101 102 LIGHT/ TNFSF14 GITR Ligand/TNFSF18 (ng/mL) Detection of LIGHT by Flow Cytometry. Expression of LIGHT/TNFSF14 on human GITR Ligand-Induced IL-8 Secretion. IL-8 secretion was induced by increasing Detection of TNF- in Mouse Macrophages. TNF- was detected in the immersion- CD4+ T cells was assessed following 36 hours of stimulation with phorbol myristate concentrations of Recombinant Human GITR Ligand (Catalog # 6987-GL) in the human fi xed RAW 263 mouse macrophage cell line treated with LPS and monensin using a acetate (PMA) (Catalog # 1201; 1 ng/mL) and calcium ionomycin. Cells were then GITR-transfected HT1080 fi brosarcoma cell line. The secreted IL-8 was measured using Goat Anti-Mouse TNF- Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # stained with an APC-conjugated Mouse Anti-Human CD4 Monoclonal Antibody the Human IL-8 DuoSet ELISA Development System (Catalog # DY208). AF-410-NA). Cells were stained with the NorthernLights™ 557-conjugated Donkey (Catalog # FAB3791A) and a PE-conjugated Mouse Anti-Human LIGHT/TNFSF14 Anti-Goat IgG Secondary Antibody (Catalog # NL001; red) and the nuclei were Monoclonal Antibody (Catalog # FAB664P; fi lled histogram) or a PE-conjugated Mouse counterstained with DAPI (blue). + IgG1 Isotype Control (Catalog # IC002P; open histogram). Cells were gated on CD4 staining cells. For additional information, please visit our website at www.RnDSystems.com/TNFSFNKCells TNF Superfamily Members Co-stimulate T Cells T cell activation requires two signals from antigen-presenting cells (APCs). The antigen-specifi c major Ligands Receptors histocompatibility complex (MHC) signal is mediated via the interaction between the antigen-MHC 4-1BB Ligand 4-1BB complex localized on the surface of APCs and the T cell receptor localized on the surface of T cells. The BAFF TACI non-antigen specifi c, co-stimulatory signal occurs via the interaction between stimulatory ligands on BAFF R CD27 Ligand the APC surface and T cell surface receptors. Several TNF superfamily members mediate T cell co- CD27 stimulation. These include 4-1BB, CD27, CD30, CD40, GITR, HVEM, DR3, and OX40 and their respective CD30 Ligand CD30 ligands, as well as TACI or BAFF R activation by BAFF, TNF RII activation by TNF-, and activation of CD40 Ligand CD40 RELT by an unknown ligand. Blockade of multiple TNF superfamily members that mediate T cell co- GITR Ligand GITR stimulation is being investigated for use in the prevention of transplant rejection.1-3 In addition, the LIGHT HVEM manipulation of GITR-mediated T cell co-stimulation is also being examined as a potential cancer TNF-E/LTD DR3 immunotherapy.4, 5 TL1A/VEGI OX40 References OX40 Ligand TNF RII 1. Dai, H. et al. (2011) Transpl. Immunol. 24:195. 4. Schaer, D.A. et al. (2012) Curr. Opin. Immunol. 24:217. RELT 2. Xiao, X. et al. (2012) J. Immunol. 188:892. 5. Avogadri, F. et al. (2011) Curr. Top. Microbiol. Immunol. TNF-D ? 3. Oura, T. et al. (2012) Am. J. Transplant. 12:1740. 344:211.

Antigen-Presenting Cell

Major Histocompatibility Complex II (MHC II) TNF Family Ligand Peptide Antigen CD4 TCR TNF Family Receptor CD3 CD3

CD3

Antigen/MHC recognition Co-stimulatory signal TNF Superfamily Members can Provide the Co-stimulatory Signal Required to Acti- vate T Cells. Naïve T cells require two stimulatory signals to become active. The fi rst signal for T cell stimulation occurs via specifi c T cell receptor binding (TCR-CD3) to MHC II-com- plexed antigen on antigen-presenting cells (APC). TNF superfamily ligands expressed on Activated T Cell the APC can provide the second co-stimulatory signal required for T cell activation by Naïve T Cell binding to their respective TNF superfamily receptors expressed on T cells.

CD27 Ligand/TNFSF7 Antibody (μg/mL) 10-2 10-1 100 101 102 50

15000 15000 Cell Proliferation (Mean CPM) 40 12000 12000 30 9000 9000 20 6000 6000 Relative Cell Number Cell Relative 10 Cell Proliferation (Mean CPM) Proliferation Cell 3000 3000

0 0 0 1 2 3 4 10-1 100 101 102 10 10 10 10 10 CD27 Ligand/TNFSF7 (μg/mL) OX40 Ligand/TNFSF4 CD27 Ligand-induced Proliferation and Neutralization by an Anti-Mouse CD27 Detection of OX40 Ligand by Flow Cytometry. Human peripheral blood Detection of CD40 on Mouse Splenocytes. CD40/TNFRSF5 was detected in Ligand Antibody. Proliferation of mouse splenic T cells was assessed following mononuclear cells treated with lipopolysaccharide and Recombinant Mouse GM-CSF immersion-fi xed mouse splenocytes using a Goat Anti-Mouse CD40/TNFRSF5 Antigen treatment with increasing concentrations of Recombinant Mouse CD27 Ligand/ (Catalog # 415-ML) were stained with a PE-conjugated Mouse Anti-Human OX40 Affi nity-purifi ed Polyclonal Antibody (Catalog # AF440). Cells were stained using a TNFSF7 (Catalog # 783-CL; orange line) in the presence of suboptimal amounts of a Ligand/TNFSF4 Monoclonal Antibody (Catalog # FAB10541P; fi lled histogram) or a PE- NorthernLights™ 557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog

Hamster Anti-Mouse CD3 Monoclonal Antibody (Catalog # MAB484). The proliferative conjugated Mouse IgG1 Isotype Control (Catalog # IC002P; open histogram). # NL001; red) and the nuclei were counterstained with DAPI (blue). eff ect induced by 10 μg/mL Recombinant Mouse CD27 Ligand/TNFSF7 was neutralized by treating the cells with increasing concentrations of a Rat Anti-Mouse CD27 Ligand/ TNFSF7 Monoclonal Antibody (Catalog # MAB783; purple line). For additional information, please visit our website at www.RnDSystems.com/TNFSFTCells TNF Superfamily Members Mediate Apoptosis TNF superfamily members mediate both the intrinsic and extrinsic pathways of caspase activation. EDAR, Fas, DR3, TNF RI, TRAIL R1, TRAIL R2, NGF R, and DR6 initiate the extrinsic pathway via recruitment Pro-apoptotic of the adaptor proteins, FADD and TRADD, and pro-caspases. TNF family receptors that do not contain death domains, including 4-1BB, CD30, HVEM, LTR, TWEAK R, and RELT, have also been shown to Ligands Receptors mediate apoptosis via indirect mechanisms such as inhibition of p38, transactivation of TNF RI, or 4-1BB Ligand 4-1BB activation-induced cell death via NF-B.1-3 Apoptosis mediated by TNF superfamily receptors is not only critical for normal developmental processes, but is also implicated in disease.4 For example, CD30 Ligand CD30 viruses encode cytokine responsive modifi ers (CRMs) that act as TNF family decoy receptors and EDA-A1 EDAR prevent apoptosis. Additionally, inhibition of TNF-mediated apoptosis, caused by down-regulation of death receptors, up-regulation of decoy receptors, or altered intracellular signaling, is associated with Fas Ligand Fas HVEM cancer development. In addition to initiating apoptosis, several TNF family members, including CD27, LIGHT BCMA, TACI, BAFF R, and OX40, inhibit apoptosis via activation of NF-B and up-regulation of anti- LTER TLA1/VEGI DR3 apoptotic proteins such as Bcl-2, Bcl-xL, and Inhibitors of Apoptosis (IAPs). The TNF receptor family also includes six decoy receptors, DcR3, DcTRAIL R1, DcTRAIL R2, TRAIL R3, TRAIL R4, and OPG, which act to TWEAK TWEAK R sequester TNF family ligands and prevent their normal physiological eff ects. References TNF-D TNF RI 1. Gullo, C. et al. (2010) PLoS One 5:e10845. 3. Krysov, S.V. et al. (2007) Mol. Cancer Ther. 6:703. TNF-E/LTD 2. Cusick, J.K. et al. (2010) Cell. Immunol. 261:1. 4. Aggarwal, B.B. et al. (2012) Blood 119:651. TRAIL R1 TL1A/VEGI TRAIL TNF-D TRAIL R2 Fas Ligand TRAIL TWEAK Neutrophins NGF R

APP DR6

? RELT DR3 Fas TRAIL R1 TNF RI TWEAK R TRAIL R2 Anti-apoptotic FADD TRADD TRADD FADD FADD FADD TRAF-2 RIP1 TRADD TRAF-2 TRAF-2 FADD CD27 Ligand CD27 TRADD FADD cIAP-1 cIAP-1 RIP1 TRAF-2 Pro-Caspase-8, -10 Daxx Pro-Caspase-8, -10 Death Pro-Caspase-8, -10 Pro-Caspase-8, -10 Domain (DD) Death Effector Domain (DED) APRIL BCMA SODD TNF Superfamily Members Initiate Caspase-Mediated Apoptosis. Several members of the TNF receptor superfamily contain cytoplasmic death domains. Upon TNF TACI superfamily ligand binding, the death domain-containing receptor recruits adaptor proteins including Fas-Associated Protein with Death Domain (FADD) and Tumor Ne- BAFF crosis Factor Receptor Type 1-Associated Death Domain (TRADD) and TNF Receptor-Associated Factor (TRAF) family members to form caspase-activating complexes. Pro- BAFF R caspases are then recruited to the complexes and activated by protease cleavage, which initiates apoptotic signaling. Silencer of Death Domains (SODD) inhibits formation of the caspase-activating complex associated with ligand bound TNF RI and blocks apoptosis. TNF family receptors that lack death domains, such as TWEAK R, can initiate OX40 apoptosis by transactivating death domain-containing TNF family receptors. OX40 Ligand

Fas Ligand/TNFSF6 Antibody (μg/mL) 10-3 10-2 10-1 100 80 1000 1000 70 Cell Viability (Mean RFU) 60 800 800 50 600 600 40 30 400 400 Relative Cell Number Cell Relative 20 (Mean RFU) Viability Cell 200 200 10

0 0 0 100 101 102 103 104 10-2 10-1 100 101 102 TRAIL R1/TNFRSF10A Fas Ligand/TNFSF6 (ng/mL) Detection of TRAIL R1 by Flow Cytometry. The HeLa human cervical cancer cell line Detection of TNF RI on Human Peripheral Blood Lymphocytes. TNF RI/TNFRSF1A Fas Ligand-induced Apoptosis and Neutralization by an Anti-Fas Ligand was stained with an APC-conjugated Mouse Anti-Human TRAIL R1/TNFRSF10A was detected in immersion-fi xed human peripheral blood lymphocytes using a Goat Antibody. Apoptosis of Jurkat cells was assessed following treatment with increasing Monoclonal Antibody (Catalog # FAB347A; fi lled histogram) or an APC-conjugated Anti-Human TNF RI/TNFRSF1A Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog concentrations of Recombinant Human Fas Ligand/TNFSF6 (Catalog # 126-FL; orange

Mouse IgG1 Isotype Control (Catalog # IC002A; open histogram). # AF225). Cells were stained with a fl uorochrome-conjugated secondary antibody line) in the presence of a cross-linking Mouse Poly-Histidine Monoclonal Antibody (red) and nuclei were counterstained (green). (Catalog # MAB050). The apoptotic eff ect induced by 2 ng/mL Recombinant Human Fas Ligand/TNFSF6 was neutralized by treating the cells with increasing concentrations of a Goat Anti-Human Fas Ligand/TNFSF6 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # AF126; purple line). For additional information, please visit our website at www.RnDSystems.com/TNFSFApoptosis TNF Superfamily Products TNF Superfamily Receptors TNF Superfamily Ligands RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs RECOMBINANT MOLECULE PROTEINS ANTIBODIES ELISAs H (E, FA, FC, IHC, WB) 4-1BB/TNFRSF9/CD137 H M M (B/N, E, FA, FC, WB) H M 4-1BB Ligand/TNFSF9 H M H (FC, WB) M (FC, WB) BAFF R/TNFRSF13C H M H (B/N, FC, WB) M (B/N, E, FC, WB) M APRIL/TNFSF13 HH (B/N, E, FC, WB) M (FC) H BCMA/TNFRSF17 H M H (B/N, E, FC, WB) M (B/N, E, FC, WB) H BAFF/BLyS/TNFSF13B H M H (B/N, FC, IHC, WB) M (B/N, FC, IHC, WB) H M CD27/TNFRSF7 H M H (B/N, FC, IHC, WB) M (E, FC, WB) M CD27 Ligand/TNFSF7 MH (FC, IHC, WB) M (B/N, E, FC, WB) M CD30/TNFRSF8 H M H (FA, FC, WB) M (E, FA, IHC, WB) M CD30 Ligand/TNFSF8 H M H (B/N, FC, WB) M (B/N, E, FC, WB) M CD40/TNFRSF5 H M H (B/N, FA, FC, IHC, WB) H M H (B/N, FC, IHC, WB) M (E, FA, FC, IF, IHC, IP, WB) CD40 Ligand/TNFSF5 H M M (B/N, E, FC, IHC, WB) H M DcR3/TNFRSF6B HH (B/N, E, IHC, WB) H EDA/Ectodysplasin H (E) H DcTRAIL R1/TNFRSF23 M (IHC, WB) EDA-A1/Ectodysplasin A1 H M DcTRAIL R2/TNFRSF22 MM (WB) EDA-A2/Ectodysplasin A2 HH (B/N, WB) DR3/TNFRSF25 H M H (FC, WB) M (IHC, WB) H (B/N, E, FC, IHC, WB) M (B/N, E, FC, WB) DR6/TNFRSF21 Fas Ligand/TNFSF6 H M R R (IHC, WB) H M H M H (E, WB) H EDA2R/TNFRSF27/XEDAR H M H (B/N, E, WB) H GITR Ligand/TNFSF18 H M H (B/N, E, FC, WB) M (B/N, E, FC, WB) H M EDAR H M H (B/N, WB) M (B/N, E, WB) M LIGHT/TNFSF14 H M H (B/N, E, FC, WB) M (FC, WB) H H (E, FC, IHC, WB) M (E, FC, IHC, WB) Lymphotoxin 12 HH (B/N) Fas/TNFRSF6/CD95 H M R F R (FC, IHC, WB) F (FC, IHC, WB) H M Lymphotoxin 21 H M H (B/N) GITR/TNFRSF18 H M H (B/N, E, FC, IHC, WB) M (E, FC, WB) H M Lymphotoxin /TNFSF3 H (WB) HVEM/TNFRSF14 H M H (E, FC, IHC, WB) M (WB) H H (B/N, FC, IHC, WB) OX40 Ligand/TNFSF4 H M M (B/N, E, FC, IHC, WB) M Lymphotoxin R/TNFRSF3 H M H (B/N, FC, WB) M (FC, IHC, WB) NGF R/TNFRSF16 H M H (FC, IHC, WB) M (IHC, WB) H M TL1A/TNFSF15 H M H (WB) M (WB) Osteoprotegerin/TNFRSF11B H M H (B/N, E, IHC, WB) M (B/N, E, IHC, WB) H M H (B/N, E, FC, IHC, WB) M (B/N, E, FC, IHC, WB) H M R B OX40/TNFRSF4 H M H (FC, WB) M (FA, FC, WB) H M R B Ca CR E F P R (B/N, E, IHC, WB) P (B/N, E, IHC, WB) Ca CR E TNF-/TNFSF1A B (E, IHC, WB) Ca (B/N, E, IHC, WB) RANK/TNFRSF11A H M H (E, FA, FC, IHC, WB) M (FA, IHC, WB) H Rb RM CR (B/N, WB) E (B/N, E, IHC, WB) F P Pr F (E, WB) (E) Rb (B/N, WB) Rb RM RELT/TNFRSF19L H M H (FC, WB) RM (B/N, E, WB) H TACI/TNFRSF13B H M (B/N, E, FC, IHC, WB) H M TNF-/TNFSF1/Lymphotoxin  H M H (B/N, E, FC, IHC, WB) M (IHC, WB) H M (B/N, E, FC, IHC, WB) TRAIL/TNFSF10 H M H (B/N, E, FC, IHC, WB) M (E, IHC, WB) H M TNFRH3/TNFRSF26 MM (FC, WB) TRANCE/TNFSF11/RANK L H M H (B/N, FC, IHC, WB) M (B/N, E, IHC, WB) M H (B/N, E, FA, FC, IHC, WB) TNF RI/TNFRSF1A H M Ca M (B/N, E, FA, FC, IHC, IP, WB) H M TWEAK/TNFSF12 H M H (B/N, E, IHC, WB) M (B/N, E, WB) H M H (B/N, E, FC, IHC, WB) TNF RII/TNFRSF1B H M M (E, FC, IHC, IP, WB) H M TNF Superfamily Regulators TRAIL R1/TNFRSF10A HH (B/N, FC, IHC, WB) RECOMBINANT TRAF-1 H (WB) MOLECULE PROTEINS ANTIBODIES TRAIL R2/TNFRSF10B H M H (FA, FC, WB) M (B/N, FC, IHC, WB) H (WB) M TRAIL R3/TNFRSF10C HH (B/N, E, FC, IHC, WB) H Daxx H (WB) TRAF-2 (WB) R (WB) FADD H (IHC, WB) TRAIL R4/TNFRSF10D HH (B/N, E, FC, IHC, WB) H H (WB) M TROY/TNFRSF19 H M H (WB) M (E, IHC, WB) M H (WB) M TRAF-3 RIP1 (WB) R (WB) (WB) R (WB) TWEAK R/TNFRSF12 H M H (B/N, IHC, WB) M (E, FC, IHC, WB) M TRAF-4 H (WB) H (WB) M SODD/BAG4 TRAF-5 H (IHC) (WB) R (WB) Proteome Profi ler™ Array Kit H (WB) M TRAF-6 H (WB) TANK (WB) KIT DESCRIPTION H (FC, IHC, TRADD WB) Human Apoptosis Array Kit Contains 4 membranes - each spotted in duplicate with 35 diff erent apoptosis antibodies: Species Key: Catalog # ARY009 Bad, Bax, Bcl-2, Bcl-x, Pro-Caspase-3, Cleaved Caspase-3, Catalase, cIAP-1, H Human, M Mouse, R Rat, B Bovine, Ca Canine, CR Cotton Rat, E Equine, F Feline, P Porcine, Pr Primate, Rb Rabbit, cIAP-2, Claspin, Clusterin, Cytochrome c, TRAIL R1/DR4, TRAIL R2/DR5, RM Rhesus Macaque, V Viral FADD, Fas/TNFRSF6, HIF-1, HO-1/HMOX1/HSP32, HO-2/HMOX2, HSP27, HSP60, HSP70, HTRA2/Omi, Livin, PON2, p21/CIP1/CDNK1A, p27/Kip1, Application Key: Phospho-p53 (S15), Phospho-p53 (S46), Phospho-p53 (S392), Phospho- B/N Blocking/Neutralization, E ELISA, FA Functional Assay, FC Flow Cytometry, IF Immunofl uorescence, Rad17 (S635), SMAC/Diablo, Survivin, TNF RI/TNFRSF1A, XIAP IHC Immunohistochemistry, IP Immunoprecipitation, WB Western blot

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Printed on recyclable paper 10% post consumer waste. NorthernLights and Proteome Profi ler are trademarks of R&D Systems. R&D Systems is a trademark of TECHNE Corporation. MA_04.12_TNF Superfamily_392

TNF Superfamily Members Mediate Other Cell Type-specifi c Responses

While many TNF superfamily members initiate apoptosis or are involved in the stimulation and homeostasis of immune cells, other TNF family members mediate alternate biological activities. Ligands Receptors Although TROY has no known ligand, both TROY and XEDAR are involved in hair follicle formation via NF-B activation. RANK, another TNF family member, initiates osteoclast diff erentiation via EDA-A2 XEDAR NF-B activation, and recent research demonstrates a role for RANK in breast cancer metastasis.1,2 The Lymphotoxin  Receptor (LTR) binds LIGHT as well as heterotrimers composed of LIGHT LTD E Lymphotoxin  and Lymphotoxin . LTR activation is necessary for the formation of secondary 1 2 LTER LTD E immune tissues, stimulation of cytokine secretion by mast cells and fi broblasts, and is also being 2 1 3 examined for its role in carcinogenesis. TWEAK mediates normal tissue remodeling and is TWEAK TWEAK R important in disease and cancer. 4,5 References TRANCE/RANK Ligand RANK 1. Nakashima, T. & Y. H. Takayanagi (2011) Ann. N. Y. Acad. Sci. 4. Michaelson, J.S. & L.C. Burkly (2009) Results Probl. Cell Diff er. 1240:E13. 49:145. TROY 2. Palafox, M. et al. (2012) Cancer Res. 72:2879. 5. Ortiz, A. et al. (2009) Cytokine Growth Factor Rev. 20:251. ? 3. Wolf, M.J. et al. (2010) Oncogene 29:5006.

RANK/TNFSF11A (μg/mL) 10-3 10-2 10-1 100 101 102 3 3

60 Osteoclast Differentiation (Mean O.D.)

50

2 2 40

30

20 1 1 Relative Cell Number Cell Relative 10 Osteoclast Differentiation (Mean O.D.) Differentiation Osteoclast

0 0 100 101 102 103 104 10-5 10-4 10-3 10-2 10-1 100 LTER/TNFRSF3 TRANCE/RANK L/TNFSF11 (μg/mL) Detection of LTR by Flow Cytometry. Human peripheral blood monocytes were TRANCE/RANK Ligand-induced Osteoclast Diff erentiation. The RAW264.7 mouse Detection of TWEAK in MDA-MB-231 Human Breast Cancer Cells. TWEAK/ stained with an APC-conjugated Mouse Anti-Human LTR/TNFRSF3 Monoclonal macrophage cell line was treated with increasing concentrations of Recombinant TNFSF12 was detected in the immersion-fi xed MDA-MB-231 human breast

Antibody (Catalog # FAB629A; fi lled histogram) or an APC-conjugated Mouse IgG1 Mouse TRANCE/RANK L/TNFSF11 (Catalog # 462-TEC; red line). Osteoclast-like cell adenocarcinoma cell line using a Goat Anti-Human TWEAK/TNFSF12 Antigen Isotype Control (Catalog # IC002A; open histogram). diff erentiation was determined by measuring tartrate-resistant acid phosphatase Affi nity-purifi ed Polyclonal Antibody (Catalog # AF1090). Cells were stained with a activity. Osteoclast diff erentiation in the presence of 10 ng/mL TRANCE/RANK NorthernLightsTM 557-conjugated Donkey Anti-Goat IgG Secondary Antibody Ligand was neutralized by treating the cells with increasing concentrations of (Catalog # NL001; red) and nuclei were counterstained with DAPI (blue). Recombinant Mouse RANK/TNFRSF11A (Catalog # 692-RK; purple line). For additional information, please visit ourwww.RnDSystems.com website at www.RnDSystems.com/TNFSFOtherFunctions