Anti traf2

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-TRAF2 is a laboratory reagent that targets the TRAF2 protein. TRAF2 is an adapter protein involved in the activation of various signaling pathways. The antibody can be used for the detection and study of TRAF2 in research applications.

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13 protocols using anti traf2

Antibody Panel for Protein Analysis

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Antibodies used in the study were as follows: anti-Flag (F3165/F7425, Sigma), anti-Myc (9B11/71D10, Cell Signaling Technology (CST)), anti-HA (16B12, Biolegend and H6908, Sigma), anti-TRAF2 (sc-136999, Santa Cruz), anti-Rab5 (sc-46692, Santa Cruz), anti-Rab7 (sc-376362, Santa Cruz), anti-Sprouty 2 (sc-100862, Santa Cruz/ab85670, Abcam), anti-EGFR (sc-373746, Santa Cruz), anti-p-Tyr (9411, CST), anti-p-Ser/Thr (ab9344, Abcam and 05-368, Sigma), anti-Ser (sc-81514, Santa Cruz), anti-Thr (sc-5267, Santa Cruz), anti-Ubiquitin (sc-8017, Santa Cruz), anti-β-Actin (AC026, Abclonal), anti-GFP (AE011, Abclonal), anti-glutathione S-transferase (GST) (2622 S, CST), anti-V5 (R960-25, Thermo Fisher and 30801ES10, Yeasen), anti-Mouse/Rabbit IgG-peroxidase secondary antibody (A0545/A9044, Sigma), anti-Mouse IgG (light chain specific)-peroxidase secondary antibody (115-005-174, Jackson), and anti-DYRK1A polyclonal antibody has been previously described [30 (link)].

Zhang P., Zhang Z., Fu Y., Zhang Y., Washburn M.P., Florens L., Wu M., Huang C., Hou Z, & Mohan M. (2021). K63-linked ubiquitination of DYRK1A by TRAF2 alleviates Sprouty 2-mediated degradation of EGFR. Cell Death & Disease, 12(6), 608.

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Immunofluorescent Analysis of Nuclear p65

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Adherent cells were fixed with methanol and incubated with anti-p65 (Santa Cruz), anti-TRAF2 (Santa Cruz) or anti-KL1 (Immunotech Biotechnologies) and then with the Alexa Fluor 488–conjugated F(ab′)2 fragment of anti-mouse IgG (Invitrogen). The slides were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Images were acquired with a Nikon Eclipse 80i fluorescence microscope (Nikon) with a 20× objective, a 377/482 nm filter and a Dxm 1200C digital camera (Nikon). They were analyzed with NIS Elements Imaging software (Nikon). The fluorescence intensity of nuclear p65 was quantified with ImageJ software (National Institutes of Health [NIH]).
Phase-contrast microscopy images were captured on a Nikon Microphot-FX microscope with a 20× objective.

Vuillier F., Gaud G., Guillemot D., Commere P.H., Pons C, & Favre M. (2014). Loss of the HPV-Infection Resistance EVER2 Protein Impairs NF-κB Signaling Pathways in Keratinocytes. PLoS ONE, 9(2), e89479.

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Evaluation of BAFF Signaling Pathway in B-cell

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CP-25 was supplied by the Chemistry Laboratory of Institute of Clinical Pharmacology of Anhui Medical University (Hefei, Anhui Province, China). Rituximab (Roche Pharma, Switzerland); Etanercept (Shanghai Guojian Pharmaceutical, Co., Ltd., China); BAFF (Pepro Tech, United States); anti-CD19-APC/PE/FITC, anti-CD20-FITC, anti-CD27-PE/APC, anti-BAFFR-APC, anti-BCMA-PE, and anti-TACI-PE (Biolegend, United States); anti-β-actin (ZSGB-BIO, China); Anti-TRAF2 (Santa Cruz, China); Anti-Phospho-NF-κBp65, anti-p100/52, and anti-Phospho-p38 MAPK (Cell Signaling Technology, United States); RPMI 1640 medium (Gibco, United States); fetal bovine serum (Zhejiang Tianhang Biotechnology, China).

Zhang F., Shu J.L., Li Y., Wu Y.J., Zhang X.Z., Han L., Tang X.Y., Wang C., Wang Q.T., Chen J.Y., Chang Y., Wu H.X., Zhang L.L, & Wei W. (2017). CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents. Frontiers in Pharmacology, 8, 933.

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Elucidating TNF-α Signaling Pathways

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Minimal essential medium-alpha (α-MEM), fetal bovine serum (FBS), and TRIzol were purchased from Invitrogen (Carlsbad, CA). Hybond C membrane and ECL Western blotting detection system were from Amersham Biosciences (Buckinghamshire, UK). Recombinant human TNF-α and the anti-TNFR1 neutralizing antibody were from R&D System (Minneapolis, MN). Luciferase assay kit was from Promega (Madison, WI). Metafectene transfection reagent was from Biontex Lab (GmbH, Planegg/Martinsried, Germany). SMARTpool RNA duplexes corresponding to Src, TRAF2, ERK2, p38 MAPK, JNK2, and scrambled #2 siRNA were from Dharmacon Research Inc (Lafayette, CO). Anti-phospho-IKKα/β, anti-phospho-NF-κB p65 (Ser⁵³⁶), anti-phospho-c-Src, anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, and anti-phospho-IκB-α antibodies were from Cell Signaling (Danver, MA). anti-NF-κB (p65), anti-lamin A, anti-TRAF2, anti-TNFR1, anti-c-Src, anti-ERK2, anti-p38, anti-JNK2, anti-IκB-α, and anti-sICAM-1 antibodies were from Santa Cruz (Santa Cruz, CA). The anti-GAPDH antibody was from Biogenesis (Boumemouth, UK). Actinomycin D (Act.D), cycloheximide (CHI), PP1, U0126, SB202190, SP600125, GM6001, MMP2/9 inhibitor, and Bay11-7082 were from Biomol (Plymouth Meeting, PA). Enzymes and other chemicals were from Sigma (St. Louis, MO).

Tsai C.L., Chen W.C., Hsieh H.L., Chi P.L., Hsiao L.D, & Yang C.M. (2014). TNF-α induces matrix metalloproteinase-9-dependent soluble intercellular adhesion molecule-1 release via TRAF2-mediated MAPKs and NF-κB activation in osteoblast-like MC3T3-E1 cells. Journal of Biomedical Science, 21(1), 12.

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Comprehensive Antibody Catalog for Protein Analysis

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Rabbit polyclonal anti-TRAF2 (#sc-876), mouse monoclonal β-actin (#sc-47778), mouse monoclonal anti-USP-14 (#sc-100630), mouse monoclonal anti-MMP-1 (#sc-58377), rabbit polyclonal anti-MMP-13 (#sc-30073), rabbit polyclonal anti-Lamin A/C (#sc-20681) and rabbit polyclonal anti-p-IkB-α (#sc-8404) antibodies were purchased from Santa Cruz Biotech, (Santa Cruz, CA). Rabbit monoclonal Anti-cIAP1 (#7065), rabbit monoclonal anti-cIAP2 (#3031), rabbit anti-USP2 (#8036), rabbit monoclonal anti-RAD23A (#24555), anti-K63 polyubiquitin (#5621), and anti-K48 polyubiquitin (#8081), anti-STAT-3 (#9132), anti-NF-κBp65 (#8242), anti-p-c-Jun (S73) (#9164), anti-p-p38 (T180/Y182) (#4511), anti-p-JNK (T183/Y185) (#9251), total JNK (#8690), total p-38 (# 9258) and p-STAT-3 (S727) (#9134) antibodies were purchased from Cell Signaling Technology (Beverly, MA). TRAF2 mouse monoclonal (#AM1895B) for immunoprecipitation were purchased from Abjent (San Diego, CA). Anti-PSMD13 (#5937-1) antibody was purchased from Epitomics (Burlingame, CA), respectively. Total ASK1 (#ab131506), p-ASK1 Thr838/845 antibodies were was purchased from Abcam (Cambridge, MA) and Cell Signaling Technology (Beverly, MA), respectively. Human Cytokine Array C5 (#AAH-CYT-5) was purchased from Ray Biotech (Norcross, GA). SMARTpool ON-TARGET plus ASK1 siRNA or negative control siRNA was purchased from GE Dharmacon (Lafayette, CO).

Akhtar N., Singh A.K, & Ahmed S. (2016). MicroRNA-17 Suppresses TNF-α Signaling by Interfering with TRAF2 and cIAP2 Association in Rheumatoid Arthritis Synovial Fibroblasts. Journal of immunology (Baltimore, Md. : 1950), 197(6), 2219-2228.

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Protein Extraction and Western Blot Analysis

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Cells were lysed in hypotonic buffer (20 mM HEPES pH 7.4, 1 mM MgCl₂, 10 mM KCl, 1 mM DTT, 1 mM EDTA pH 8.0) containing the protease inhibitor mixture (Sigma). After sonication and sitting on ice for 20 min, the lysates were centrifuged at 14,000×g for 30 min at 4°C. Protein concentrations were determined using the Bio-Rad protein assay kit, with albumin as standards. Total proteins (25 µg) were electrophoresed on 10% SDS-polyacrylamide gel and transferred onto nitrocellulose membrane (Schleicher and Schuell) by reverse electrophoresis. After being blocked, the membrane was stained with anti-FBXO7 (1∶3000 dilution, Abnova), anti-TRAF2 (1∶500 dilution, Santa Cruz), anti-neomycin (1∶1000 dilution, Millipore), anti-tubulin (1∶10000 dilution, GeneTex), anti-GAPDH (1∶1000 dilution, MDBio), anti-actin (1∶10000 dilution, Millipore), anti-EGFP (1∶200 dilution, Santa Cruz), or anti-ubiquitin (1∶1000 dilution, Dako) antibody. The immune complexes were detected using horseradish peroxidase-conjugated goat anti-mouse (Jackson ImmunoResearch) or goat anti-rabbit (Rochland) IgG antibody (1∶10000 dilution) and Immobilon Western Chemiluminescent HRP substrate (Millipore).

Chen C.M., Chen I.C., Huang Y.C., Juan H.F., Chen Y.L., Chen Y.C., Lin C.H., Lee L.C., Lee C.M., Lee-Chen G.J., Lai Y.J, & Wu Y.R. (2014). FBXO7 Y52C Polymorphism as a Potential Protective Factor in Parkinson's Disease. PLoS ONE, 9(7), e101392.

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Immunoblotting Reagents for Cell Death Signaling

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Antibodies and other reagents were obtained as follows: anti-AF6 and anti-RIPK1(receptor-interacting serine/threonine kinase 1) antibodies from BD Transduction Laboratories; anti-pRIPK1(S166), anti-TNFR1(TNF receptor superfamily member 1A), anti-Hsp90(heat shock protein 90), and anti-GAPDH(glyceraldehyde-3-phosphate dehydrogenase) antibodies from Cell Signaling Technology; anti-pRIPK3(Ser232) (receptor-interacting serine/threonine kinase 3), anti-pMLKL(Ser345)(mixed lineage kinase domain-like pseudokinase), anti-MLKL and anti-CYLD(CYLD lysine 63 deubiquitinase) antibodies from abcam; anti-USP21, anti-FADD (Fas-associated via death domain), anti-TRAF2(TNF receptor-associated factor 2), and anti-TRADD(TNFRSF1A associated via death domain) antibodies from Santa Cruz Biotechnology; anti-A20(TNF alpha-induced protein 3) antibody from ABClonal; anti-RIPK3 from Prosci; and anti-actin antibody from Servicebio. Smac was a gift from Haibing Zhang’s lab. Other reagents were purchased as follows: mouse TNFα (Minneapolis), lipopolysaccharide (LPS) (Sigma-Aldrich Cat. L4516), Nec-1 (Enzo Life Science), Z-Val-Ala-Asp (OMe)-FMK (z-VAD) and Bv6 (MedChemExpress).

Xinyu W., Qian W., Yanjun W., Jingwen K., Keying X., Jiazheng J., Haibing Z., Kai W., Xiao X, & Lixing Z. (2023). Polarity protein AF6 functions as a modulator of necroptosis by regulating ubiquitination of RIPK1 in liver diseases. Cell Death & Disease, 14(10), 673.

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Western Blot Analysis of Liver Proteins

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Primary hepatocytes and mouse liver specimens were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4; 1% Triton X-100; 25 mM HEPES; 150 mM NaCl; 0,2% SDS; 5 mM MgCl₂; 1 mM Na₃VO₄; 1 mM NaF and proteases inhibitors (Roche, #04 693 132 001)), and liver were crushed with UltraTurax. After 40 min in ice, samples were centrifuged at 13 000 g. Proteins from supernatant were assayed with the Bradford method (Bio-Rad). Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were blocked with non-fat milk or BSA 3–5% in TBS (20 mM Tris, 137 mM NaCl) during 1–2 h and incubated overnight with primary antibody anti-cleaved-caspase-3, anti-RIPK1 (Cell Signaling) anti-actin (Sigma A3854), anti-TRAF2 (Santa Cruz Biotechnology, sc-876), anti-phospho-IκBα (Cell Signaling, #2859) or anti-IκBα (Santa Cruz Biotechnology, sc-371) at 4 °C, and then with secondary goat anti-rabbit immunoglobulins/HRP (Dako, P0448). Protein–antibody complexes were revealed by enhanced chemiluminescence (Millipore, Billerica, MA, USA) and ImageQuant LAS-4000 mini imager analysis (GE Healthcare, Chicago, IL, USA).

Filliol A., Piquet-Pellorce C., Le Seyec J., Farooq M., Genet V., Lucas-Clerc C., Bertin J., Gough P.J., Dimanche-Boitrel M.T., Vandenabeele P., Bertrand M.J, & Samson M. (2016). RIPK1 protects from TNF-α-mediated liver damage during hepatitis. Cell Death & Disease, 7(11), e2462-.

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Immunoblot analysis of RANKL signaling

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OSCC cells were seeded (5 × 10⁵/well) in 6-well plates and supplemented with DMEM containing 10% FBS. Cells were stimulated with or without RANKL or OPG as noted. Total cell lysates were prepared in a buffer containing 20 mM Tris-HCl at pH 7.4, 1% Triton X-100, 1 mM EDTA, 1.5 mM MgCl_2, 10% glycerol, 150 mM NaCl, 0.1 mM Na₃VO₄ and 1× protease inhibitor cocktail. The protein content of the samples was measured using the Bradford protein assay reagent (Bio-Rad, Hercules, CA). Protein (100 μg) samples were subjected to SDS–PAGE (4–15% Tris-HCl gels) and blot transferred on to a PVDF membrane. Immunoblotting was performed with anti-RANKL (Santa Cruz, SC377079), anti-proliferating cell nuclear antigen (PCNA) (Santa Cruz, SC-25280), anti-TRAF2 (Santa Cruz, SC-136999), anti-TRAF6 (Santa Cruz, SC-8409), anti-IKKα/β (Santa Cruz, SC-101706), anti-NFATc2 (Santa Cruz, SC-13034), anti-p-IkB-α (Cell Signaling Technology, Danvers, MA, cat #2859), anti-IKB-α (Cell Signaling Technology, cat#4814), and anti-β-actin (Sigma–Aldrich, Cat #A1978) antibodies. The bands were detected using the enhanced chemiluminescence (ECL) detection system and quantified by densitometric analysis using the NIH ImageJ Program.

Sambandam Y., Ethiraj P., Hathaway-Schrader J.D., Novince C.M., Panneerselvam E., Sundaram K, & Reddy S.V. (2018). Autoregulation of RANK ligand in oral squamous cell carcinoma tumor cells. Journal of cellular physiology, 233(8), 6125-6134.

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Inflammatory Signaling Pathway Analysis

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Anti-β-actin, anti-ICAM-1, anti-p42, anti-JNK2, anti-TNFR1, anti-c-Jun, and anti-TRAF2 antibodies were from Santa Cruz (Santa Cruz, CA). Anti-phospho-PKCδ, anti-phospho-p42/p44 MAPK, anti-phospho-c-Jun, and anti-phospho-JNK1/2 antibodies were from Cell Signaling (Danver, MA). Ro318220, Rottlerin, Gö6976, U0126, SB202190, SP600125, and Tanshinone IIA were from Biomol (Plymouth Meetings, PA). Anti-TNFR neutralizing antibody, anti-ICAM-1 neutralizing antibody, and TNF-α were from R&D Systems (Minneapolis, MN). All other reagents were from Sigma (St. Louis, MO).

Lee I.T., Liu S.W., Chi P.L., Lin C.C., Hsiao L.D, & Yang C.M. (2015). TNF-α Mediates PKCδ/JNK1/2/c-Jun-Dependent Monocyte Adhesion via ICAM-1 Induction in Human Retinal Pigment Epithelial Cells. PLoS ONE, 10(2), e0117911.

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