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Traf6

Manufactured by Santa Cruz Biotechnology
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TRAF6 is a laboratory reagent that functions as an E3 ubiquitin-protein ligase. It is involved in the activation of the NF-kB transcription factor and the regulation of various cellular processes. The core function of TRAF6 is to facilitate the transfer of ubiquitin to target proteins, thereby modulating their activity and cellular signaling pathways.

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Lab products found in correlation

Irak1, by Santa Cruz Biotechnology (5 mentions) β actin, by Merck Group (5 mentions) Gapdh, by Cell Signaling Technology (5 mentions) Gapdh, by Santa Cruz Biotechnology (4 mentions) Pvdf membrane, by Merck Group (4 mentions) Flag tag (flag), by Merck Group (3 mentions) Myd88, by Santa Cruz Biotechnology (3 mentions) P jnk, by Cell Signaling Technology (3 mentions) C fos, by Santa Cruz Biotechnology (3 mentions) Nfatc1, by Santa Cruz Biotechnology (3 mentions) β actin, by Proteintech (3 mentions) Ubiquitin, by Santa Cruz Biotechnology (3 mentions) Protein a g plus beads, by Santa Cruz Biotechnology (3 mentions) Nf κb, by Santa Cruz Biotechnology (3 mentions) Pt183 y185 jnk, by Cell Signaling Technology (2 mentions) Pt202 y204 erk, by Cell Signaling Technology (2 mentions) Ps32 36 iκbα, by Cell Signaling Technology (2 mentions) Ps176 180 ikkα β, by Cell Signaling Technology (2 mentions) Py416 c src, by Cell Signaling Technology (2 mentions) Pt180 y182 p38, by Promega (2 mentions)

47 protocols using traf6

1

Protein Fractionation and Immunoprecipitation Analysis

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Cells were lysed in cold RIPA buffer in the presence of PMSF, sodium orthovanadate, protease and phosphatase inhibitors. Protein concentration was evaluated by a BCA assay (32106, Pierce). For cytosol and nuclei protein fractionation, cytosol proteins were recovered in 10 mM Hepes pH 7.9, 10 mM KCl, 0,1 mM EDTA, 0,1 mM EGTA, 0,4 % NP40, 1 mM DTT, 0,1 mM PMSF, 10 µg/ml aprotonin, 1 mM OVA. For immunoprecipitation, TRAF6, hnRNPA1 or M2 antibodies (2µg) were added to cell lysates (1–5 mg) for 3 h at 4°C and captured by the addition of Protein A/G Plus beads (sc-2003; Santa Cruz) as described before51 (link). The immune complexes were recovered and detected by immunoblotting. Western blot analysis was performed with the following antibodies: TRAF6 (sc-7221; Santa Cruz)52 , ubiquitin (sc-8017; Santa Cruz), Myc-tag (2278; Cell Signaling), M2 (F3105; Sigma), Arhgap1/Cdc42gap (discontinued; BD Laboratories)29 (link),53 (link), Cdc42 (ab64533; Abcam), hnRNPA1 (sc-10030; Santa Cruz), Tubulin (ab6160; Abcam), β-actin (4968; Cell Signaling), GAPDH (5174; Cell Signaling).
Fang J., Bolanos L., Choi K., Liu X., Christie S., Akunuru S., Kumar R., Wang D., Chen X., Greis K.D., Stoilov P., Filippi M.D., Maciejewski J.P., Garcia-Manero G., Weirauch M.T., Salamonis N., Geiger H., Zheng Y, & Starczynowski D.T. (2016). Ubiquitination of the spliceosome auxiliary factor hnRNPA1 by TRAF6 links chronic innate immune signaling with hematopoietic defects and myelodysplasia. Nature immunology, 18(2), 236-245.
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2

Protein Fractionation and Immunoprecipitation Analysis

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Cells were lysed in cold RIPA buffer in the presence of PMSF, sodium orthovanadate, protease and phosphatase inhibitors. Protein concentration was evaluated by a BCA assay (32106, Pierce). For cytosol and nuclei protein fractionation, cytosol proteins were recovered in 10 mM Hepes pH 7.9, 10 mM KCl, 0,1 mM EDTA, 0,1 mM EGTA, 0,4 % NP40, 1 mM DTT, 0,1 mM PMSF, 10 µg/ml aprotonin, 1 mM OVA. For immunoprecipitation, TRAF6, hnRNPA1 or M2 antibodies (2µg) were added to cell lysates (1–5 mg) for 3 h at 4°C and captured by the addition of Protein A/G Plus beads (sc-2003; Santa Cruz) as described before51 (link). The immune complexes were recovered and detected by immunoblotting. Western blot analysis was performed with the following antibodies: TRAF6 (sc-7221; Santa Cruz)52 , ubiquitin (sc-8017; Santa Cruz), Myc-tag (2278; Cell Signaling), M2 (F3105; Sigma), Arhgap1/Cdc42gap (discontinued; BD Laboratories)29 (link),53 (link), Cdc42 (ab64533; Abcam), hnRNPA1 (sc-10030; Santa Cruz), Tubulin (ab6160; Abcam), β-actin (4968; Cell Signaling), GAPDH (5174; Cell Signaling).
Fang J., Bolanos L., Choi K., Liu X., Christie S., Akunuru S., Kumar R., Wang D., Chen X., Greis K.D., Stoilov P., Filippi M.D., Maciejewski J.P., Garcia-Manero G., Weirauch M.T., Salamonis N., Geiger H., Zheng Y, & Starczynowski D.T. (2016). Ubiquitination of the spliceosome auxiliary factor hnRNPA1 by TRAF6 links chronic innate immune signaling with hematopoietic defects and myelodysplasia. Nature immunology, 18(2), 236-245.
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3

Immunoblotting, Co-IP, and Recombinant Protein Purification

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Experimental procedures of immunoblotting, co-immunoprecipitation, and purification of recombinant glutathione-S-transferase (GST)-tagged or non-tagged proteins were performed as described previously [19 (link), 34 (link)]. Immunoblotting was performed using antibody specific to human TRIP6, A20 (Bethyl Laboratories), pT183/Y185-JNK, pT202/Y204-ERK, pS32/36-IκBα, pS176/180-IKKα/β, pY416-c-Src, CYLD, IκBα, IKKβ, Histone H3 (Cell Signaling, Danvers, MA, USA), pT180/Y182-p38 (Promega, Madison, WI, USA), ERK, JNK, mouse TRIP6 (BD Biosciences, San Jose, CA, USA), HA (Cayman, Ann Arbor, MI, USA), FLAG (Sigma-Aldrich), GFP, TRAF6, p38, c-Src, GAPDH, GST, ubiquitin, or phosphotyrosine (Santa Cruz Biotechnology, Dallas, TX, USA).
Lin F.T., Lin V.Y., Lin V.T, & Lin W.C. (2016). TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6 to promote the LPA2 receptor-mediated TRAF6 activation. Cell Discovery, 2, 15048-.
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4

Immunoblot Analysis of Signaling Proteins

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Three days after the third vaccination, splenocytes from immunized C57BL/6 and HBV-transgenic mice were lysed and centrifuged. The supernatants were mixed with Laemmli loading buffer and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene fluoride membranes (Millipore Corporation, Billerica, USA), and blotted with primary antibodies. Subsequently, membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized by enhanced chemiluminescence (Pierce, Appleton, WI). Antibodies against MyD88, IRAK1, TRAF6, phospho-IκB and phospho-NF-κB were purchased from Santa Cruz Biotechnology, Inc (Texas).
He M., Su D., Liu Q., Gao W, & Kang Y. (2017). Mushroom lectin overcomes hepatitis B virus tolerance via TLR6 signaling. Scientific Reports, 7, 5814.
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5

Western Blotting Protein Analysis

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Western blotting was performed according to the previous study (Liu et al., 2018 (link)). The antibodies used were shown as follows: TRAF6 (sc-8409,1:500, Santa Cruz), p-NF-κB (3033, 1:1000, Cell Signaling, Danvers, MA, United States), p-ERK (9101, 1:1000, Cell Signaling), p-JNK (4688,1:1000, Cell Signaling), p-p38 (9211,1:1000, Cell Signaling), and GAPDH (MAB374,1:10,000, Millipore, Billerica, MA, United States).
Huang H., Xia A., Sun L., Lu C., Liu Y., Zhu Z., Wang S., Cai J., Zhou X, & Liu S. (2021). Pathogenic Functions of Tumor Necrosis Factor Receptor-Associated Factor 6 Signaling Following Traumatic Brain Injury. Frontiers in Molecular Neuroscience, 14, 629910.
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6

Antibodies and Reagents for NF-κB Signaling

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Polyclonal antibodies generated against DREAM, ICAM-1, USF1, TRAF2, TRAF6, NF-κB1, NF-κB2, RelB, c-Rel, and IκBα were obtained from Santa Cruz Biotechnology, Inc. Anti-A20 mouse monoclonal antibody (mAb; 59A426) was from Calbiochem. c-Fos polyclonal antibody (pAb), phospho-TAK1 (Thr184/187) pAb, TAK1 rabbit mAb (D94D7), phospho-p38 MAPK (Thr/Tyr182) mAb (28B10), p38 MAPK pAb, phospho-SAPK/JNK (Thr183/Tyr185) rabbit mAb (81E11), SAPK/JNK rabbit mAb (56G8), IKKα pAb, phospho-IKKα/β (Ser176/180) pAb, IKKβ pAb, phospho-IκBα (Ser32) rabbit mAb (14D4), RIP1 pAb, and RIP2 pAb were from Cell Signaling. IKKγ mAb (clone 72C627) and DREAM mAb (clone 40A5) were obtained from Upstate. p65/RelA pAb was from Chemicon. Control siRNA was from Qiagen and hUSF1-siRNA (SMARTpool, cat # L003617) was from Dharmacon. siRNA transfection reagent was obtained from Santa Cruz Biotechnology, Inc. Lipids (dimethyldioctadecylammonium bromide and chlolesterol) for liposome preparation and anti-β-actin mAb (clone AC-15) were purchased from Sigma. PCR primers were custom-synthesized from Integrated DNA Technologies.
Tiruppathi C., Soni D., Wang D.M., Xue J., Singh V., Thippegowda P.B., Cheppudira B.P., Mishra R.K., DebRoy A., Qian Z., Bachmaier K., Zhao Y., Christman J.W., Vogel S.M., Ma A, & Malik A.B. (2014). The transcription factor DREAM represses A20 and mediates inflammation. Nature immunology, 15(3), 239-247.
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7

Osteoclast IFN-γ Response Signaling

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Osteoclasts were cultured with or without IFN-γ (10 ng/ml) for 2 hours. Cells were lysed with RIPA buffer (Sigma-Aldrich) containing 0.1mM PMSF, 1× EDTA-free protease inhibitor cocktail (Calbiochem), 1× Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich). Total protein concentration was determined by DC Protein Assay (Bio-Rad Laboratories), and equal amounts of total protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The primary antibodies included anti-SOCS-1 (Cell Signaling Technology), TRAF6 (Santa Cruz Biotechnology) and anti–β actin (Thermo Scientific).
Lafferty M.K., Fantry L., Bryant J., Jones O., Hammoud D., Weitzmann M.N., Lewis G.K., Garzino-Demo A, & Reid W. (2014). Elevated Suppressor of Cytokine Signaling-1 (SOCS-1): A Mechanism for Dysregulated Osteoclastogenesis in HIV Transgenic Rats. Pathogens and disease, 71(1), 81-89.
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8

Detecting K48 and K63 Polyubiquitination

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The cell lysates were prepared, and 10 µg of TRAF6 (Santa Cruz Biotechnology # sc‐8409) antibodies and 1 mg of each protein sample were used for co‐IP assays (Thermo Scientific, USA) to detect K48‐specific (1:1000, Abcam # ab140601) and K63‐specific (1:1000, Abcam # ab179434) polyubiquitination. The eluted protein samples analysed by Western blot.
Yan K., Wu C., Ye Y., Li L., Wang X., He W., Ren S, & Xu Y. (2020). A20 inhibits osteoclastogenesis via TRAF6‐dependent autophagy in human periodontal ligament cells under hypoxia. Cell Proliferation, 53(3), e12778.
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9

Western Blot Analysis of Inflammatory Signaling

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Western blot assay was performed as described in our previous study20 (link). Briefly, the protein extracts were heated with the sample buffer for 10 mins, followed by divided on a 10% polyacrylamide gel, and then transferred into the PVDF membrane. The membranes were blocked with 5% BSA, followed by maintained with primary antibodies for MyD88 (1:1000, Cell Signaling Technology), IRAK1 (1:2000, Cell Signaling Technology) and TRAF6 (1:1000, Santa Cruz Biotechnology). In addition, antibodies against IL-1β (1:2000, Santa Cruz Biotechnology), IL-6 (1:1000, Cell Signaling Technology) and TNF-α (1:2000, Santa Cruz Biotechnology) were used. Then the membranes were rinsed in TBST to be incubated with corresponding secondary antibodies at room temperature for 1 h. β-actin was used to act as a loading control. LI-COR Odyssey System was used to vision the protein bands in the membranes.
Wang L.Q, & Zhou H.J. (2018). LncRNA MALAT1 promotes high glucose-induced inflammatory response of microglial cells via provoking MyD88/IRAK1/TRAF6 signaling. Scientific Reports, 8, 8346.
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10

Molecular Mechanisms of Osteoclast Differentiation

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Cyclophosphamide (CPD) was purchased from Shengdi Company (Jiangsu, China). M-CSF and RANKL were purchased from Peprotech (New Jersey, USA). Primary antibodies against active β-catenin, Runx2, Cyclin D1, c-Myc, IκBα, phospho-IκBα, JNK, phospho-JNK, p38 and phospho-p38 were purchased from Cell Signaling Technology (Beverly, MA, USA) and others against TRAF-6, TRAF-3, NFATc1, RANK and c-Fos were purchased from Santa Cruz Biotechnology (CA, USA). The antibodies against β-catenin, phospho-Gsk-3β, and DKK1 were purchased from Abcam Company (Cambridge, UK).
Zhao D., Wang C., Zhao Y., Shu B., Jia Y., Liu S., Wang H., Chang J., Dai W., Lu S., Shi Q., Yang Y., Zhang Y, & Wang Y. (2017). Cyclophosphamide causes osteoporosis in C57BL/6 male mice: suppressive effects of cyclophosphamide on osteoblastogenesis and osteoclastogenesis. Oncotarget, 8(58), 98163-98183.
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