TRAF-2 Antibody (324522) [Unconjugated]

Western blot shows lysates of Raji human Burkitt's lymphoma cell line, SW3T3 mouse contact inhibited fibroblast cell line, and L6 rat myoblast cell line. PVDF membrane was probed with 0.25 µg/mL Mouse Anti-Human/Mouse/Rat TRAF-2 Monoclonal Antibody (Catalog # MAB3277) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). For additional reference, recombinant human TRAF-1, -2, -3, -4, -5, and -6 (2 ng/lane) were included. A specific band for TRAF-2 was detected at approximately 56 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 2.

TRAF-2 was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line using Mouse Anti-Human/Mouse/Rat TRAF-2 Monoclonal Antibody (Catalog # MAB3277) at 3 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Western blot shows lysates of HEK293T human embryonic kidney parental cell line and TRAF-2 knockout HEK293T cell line (KO). PVDF membrane was probed with 0.25 µg/mL of Mouse Anti-Human/Mouse/Rat TRAF-2 Monoclonal Antibody (Catalog # MAB3277) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for TRAF-2 at approximately 62 kDa (as indicated) in the parental HEK293T cell line, but is not detectable in knockout HEK293T cell line. GAPDH (Catalog # MAB5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

RAR gamma is required for TNF-induced RIP1-dependent apoptosis and necroptosis. a Cell death analysis of HT-29 clones cont-shRNA, RAR gamma -shRNA-A, RAR gamma -shRNA-B, RAR gamma -shRNA-C or RIP1-shRNA when treated with TSZ for 24 h was determined by PI staining using flow cytometry (left panel) (*P < 0.05 versus cont-shRNA; ANOVA). The bars represent the mean ± s.e.m. of three experiments. Western blot analysis of cells as mentioned in left panel; cell lysates were probed with antibodies as indicated (right panel). b Cell death determination by PI staining using flow cytometry of HT-29 cont-shRNA, RAR gamma -shRNA-A or RAR gamma -shRNA-A cells reconstituted with RAR gamma -shRNA resistant RAR gamma protein (RAR gamma -shRNA-A-rRAR gamma ) when treated with DMSO or TSZ for 24 h (left panel) (*P < 0.05 versus cont-shRNA. #P < 0.05 versus RAR gamma -shRNA-A; ANOVA). The bars represent the mean ± s.e.m. of three experiments. Western blot analysis of cells as mentioned in left panel; cell lysates were probed with antibodies as indicated (right panel). c, d HT-29, RAR gamma -shRNA-A, or RIP1-shRNA cells were treated with necrotic (DMSO, TS, or TSZ) c or apoptotic (CHX, TC or TCZ) d conditions for 24 h. PI positive population was determined by flow cytometry. (*P < 0.05 versus cont-shRNA; ANOVA). The bars represent the mean ± s.e.m. of three experiments. e Western blot analysis of HT-29 cont-shRNA cells infected with TRADD-shRNA, or RAR gamma -shRNA-A cells infected with TRADD-shRNA lentivirus (RAR gamma -shRNA-A + TRADD-shRNA). Cell lysates were probed with antibodies as indicated. f Cells from e were treated with DMSO, TS, or TSZ for 24 h and the PI-positive population was determined by flow cytometry. (*P < 0.05 versus cont-shRNA; ANOVA). The bars represent the mean ± s.e.m. of three experiments. All blots above are representative of one of three experiments Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/28871172), licensed under a CC-BY license. Not internally tested by R&D Systems.

Analysis of the activity of CD40 related proteins involved in transduction leading to activation of key inflammatory/survival pathway and transcription factors. (A, B) Immature dendritic cells were differentiated from monocytes derived from PBMCs donors and treated with the indicated treatments over 4 hours. (A) Representative western blot analysis (n=3-6) of CD40 related signaling adaptor proteins and related inflammatory/survival pathway and transcription factors using beta-actin as a loading control. HERA-CD40L treatment is highlighted by purple rectangles shaded by timepoints. (B) Representative subcellular fractionation and western blot analysis 15 minutes after treatment (n=3), the indicated subcellular fractions are determined by cytosolic, RIPA soluble and insoluble samples, as indicated. RIPA samples correspond to membrane and nuclear fractions while RIPA insoluble samples correspond to insoluble lipid rafts, nuclear insoluble fraction, and bound nuclear chromatin. (C) Dendritic cell activation surface marker expression analysis and IL-12 production comparison between the indicated treatments. Data displayed as mean +/- SEM (n=3 donors), Standard unpaired t-test comparison. Statistics are represented as either not significant (ns), p-value = <0.05 (*), or p-value = <0.005 (***). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37304285), licensed under a CC-BY license. Not internally tested by R&D Systems.

Analysis of the activity of CD40 related proteins involved in transduction leading to activation of key inflammatory/survival pathway and transcription factors. (A, B) Immature dendritic cells were differentiated from monocytes derived from PBMCs donors and treated with the indicated treatments over 4 hours. (A) Representative western blot analysis (n=3-6) of CD40 related signaling adaptor proteins and related inflammatory/survival pathway and transcription factors using beta-actin as a loading control. HERA-CD40L treatment is highlighted by purple rectangles shaded by timepoints. (B) Representative subcellular fractionation and western blot analysis 15 minutes after treatment (n=3), the indicated subcellular fractions are determined by cytosolic, RIPA soluble and insoluble samples, as indicated. RIPA samples correspond to membrane and nuclear fractions while RIPA insoluble samples correspond to insoluble lipid rafts, nuclear insoluble fraction, and bound nuclear chromatin. (C) Dendritic cell activation surface marker expression analysis and IL-12 production comparison between the indicated treatments. Data displayed as mean +/- SEM (n=3 donors), Standard unpaired t-test comparison. Statistics are represented as either not significant (ns), p-value = <0.05 (*), or p-value = <0.005 (***). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37304285), licensed under a CC-BY license. Not internally tested by R&D Systems.

Analysis of the activity of CD40 related proteins involved in transduction leading to activation of key inflammatory/survival pathway and transcription factors. (A, B) Immature dendritic cells were differentiated from monocytes derived from PBMCs donors and treated with the indicated treatments over 4 hours. (A) Representative western blot analysis (n=3-6) of CD40 related signaling adaptor proteins and related inflammatory/survival pathway and transcription factors using beta-actin as a loading control. HERA-CD40L treatment is highlighted by purple rectangles shaded by timepoints. (B) Representative subcellular fractionation and western blot analysis 15 minutes after treatment (n=3), the indicated subcellular fractions are determined by cytosolic, RIPA soluble and insoluble samples, as indicated. RIPA samples correspond to membrane and nuclear fractions while RIPA insoluble samples correspond to insoluble lipid rafts, nuclear insoluble fraction, and bound nuclear chromatin. (C) Dendritic cell activation surface marker expression analysis and IL-12 production comparison between the indicated treatments. Data displayed as mean +/- SEM (n=3 donors), Standard unpaired t-test comparison. Statistics are represented as either not significant (ns), p-value = <0.05 (*), or p-value = <0.005 (***). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37304285), licensed under a CC-BY license. Not internally tested by R&D Systems.

Analysis of the activity of CD40 related proteins involved in transduction leading to activation of key inflammatory/survival pathway and transcription factors. (A, B) Immature dendritic cells were differentiated from monocytes derived from PBMCs donors and treated with the indicated treatments over 4 hours. (A) Representative western blot analysis (n=3-6) of CD40 related signaling adaptor proteins and related inflammatory/survival pathway and transcription factors using beta-actin as a loading control. HERA-CD40L treatment is highlighted by purple rectangles shaded by timepoints. (B) Representative subcellular fractionation and western blot analysis 15 minutes after treatment (n=3), the indicated subcellular fractions are determined by cytosolic, RIPA soluble and insoluble samples, as indicated. RIPA samples correspond to membrane and nuclear fractions while RIPA insoluble samples correspond to insoluble lipid rafts, nuclear insoluble fraction, and bound nuclear chromatin. (C) Dendritic cell activation surface marker expression analysis and IL-12 production comparison between the indicated treatments. Data displayed as mean +/- SEM (n=3 donors), Standard unpaired t-test comparison. Statistics are represented as either not significant (ns), p-value = <0.05 (*), or p-value = <0.005 (***). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37304285), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Hu, Mu, Rt
• Applications: WB, ICC/IF, KO
• Clone: 324522
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Concentration: LYOPH

TRAF-2 Antibody (324522) [Unconjugated] Summary

• Immunogen: E. coli-derived recombinant human TRAF-2
  Met1-Leu501
  Accession # Q12933
• Specificity: Detects human, mouse, and rat TRAF-2 in direct ELISAs and Western blots. In direct ELISAs and Western blots, no cross-reactivity with recombinant human TRAF-1, -3, -4, -5, or -6 is observed.
• Source: N/A
• Isotype: IgG2a
• Clonality: Monoclonal
• Host: Mouse
• Gene: TRAF2
• Purity Statement: Protein A or G purified from hybridoma culture supernatant

Applications/Dilutions

• Dilutions:
  • Immunocytochemistry 3-25 ug/mL
  • Knockout Validated
  • Western Blot 0.25 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for TRAF-2 Antibody (324522) [Unconjugated]

• E3 ubiquitin-protein ligase TRAF2
• EC 6.3.2.-
• MGC:45012
• TNF receptor-associated factor 2
• TRAF2
• TRAF-2
• TRAP3
• TRAP3TRAP
• tumor necrosis factor type 2 receptor associated protein 3
• Tumor necrosis factor type 2 receptor-associated protein 3

Background

Tumor Necrosis Factor (TNF) Receptor-Associated Factors (TRAFs) are a family of adaptor proteins that interact with a wide range of cell surface receptors and participate in the regulation of cell survival, proliferation, differentiation, and stress response. TRAFs were identified by their ability to form complexes with TNF receptor superfamily members but more recently are reported to also bind to Toll/IL-1 receptor family members and mediate cellular signaling. Six members of the TRAF family have been identified. All TRAF proteins have a homologous C-terminal TRAF domain that can bind the cytoplasmic domain of receptors as well as other TRAFs. TRAFs 2‑6 have N-terminal RING and zinc finger domains that are involved in signaling downstream events. TRAF-2, also known as TNF Receptor-Associated Protein (TRAP), is a 501 amino acid, 56 kDa protein which interacts with the cytoplasmic domain of TNFR1, TNFR2 and CD40 to mediate the activation of NF-kappa-B and AP-1 family of transcription factors. TRAF-2 can self-associate as well as form heterodimers with TRAF-1.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for TRAF-2 Antibody (MAB3277)(6)

Publications using MAB3277

• De Luca, A;Faienza, F;Fulci, C;Nicolai, E;Calligari, P;Palumbo, C;Caccuri, AM; Molecular and cellular evidence of a direct interaction between the TRAF2 C-terminal domain and ganglioside GM1 The international journal of biochemistry & cell biology 2023-12-22 [PMID: 38142771] (Functional Assay, Human)
• Jamie Frankish, Debayan Mukherjee, Erminia Romano, Katharina Billian-Frey, Matthias Schröder, Karl Heinonen et al. The CD40 agonist HERA-CD40L results in enhanced activation of antigen presenting cells, promoting an anti-tumor effect alone and in combination with radiotherapy Frontiers in Immunology 5/26/2023 [PMID: 37304285]
• Fang , Di-Feng, He , Kun, Wang , Na, Sang , Zhi-Hong, Qiu , Xin, Xu , Guang, Jian , Zhao, Liang , Bing, Li , Tao, Li , Hui-Yan, Li , Ai-Ling, Zhou , Tao, Gong , Wei-Li, Yang , Baoli, Karin , Michael, Zhang , Xue-Min, Li , Wei-Hua NEDD4 ubiquitinates TRAF3 to promote CD40-mediated AKT activation. Nat Commun, 2014-07-29;5(0):4513. 2014-07-29 [PMID: 25072696] (Western Blot, Human, Mouse)
• Jin HR, Jin X, Lee JJ Zinc-finger protein 91 plays a key role in LIGHT-induced activation of non-canonical NF-kappaB pathway. Biochem. Biophys. Res. Commun., 2010-09-06;400(4):581-6. 2010-09-06 [PMID: 20804734] (Western Blot, Human)
• Kolliputi N, Waxman AB IL-6 cytoprotection in hyperoxic acute lung injury occurs via suppressor of cytokine signaling-1-induced apoptosis signal-regulating kinase-1 degradation. Am. J. Respir. Cell Mol. Biol., 2008-09-05;40(3):314-24. 2008-09-05 [PMID: 18776134] (Western Blot, Human)
• Pobezinskaya YL, Kim YS, Choksi S, Morgan MJ, Li T, Liu C, Liu Z The function of TRADD in signaling through tumor necrosis factor receptor 1 and TRIF-dependent Toll-like receptors. Nat. Immunol., 2008-07-20;9(9):1047-54. 2008-07-20 [PMID: 18641653] (Western Blot, Mouse)

Bioinformatics

• Gene Symbol: TRAF2
• Uniprot:
  • Q12933()