Human TAK1 Antibody

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	-Mouse (1:1000)

Protocol tips
-Mouse (1:1000)

Publication protocol

We lysed cells in RIPA buffer (10 mM Tris-HCL, 1 mM EDTA, 1% sodium dodecyl sulfate [SDS], 1% Nonidet P-40, 1: 100 proteinase inhibitor cocktail, 50 mM β-glycerophosphate, 50 mM sodium fluoride). We then separated lysates on a 10% SDS polyacrylamide gel and transferred to membranes by a semi-dry transfer apparatus (Bio-Rad). We blocked membranes with 5% milk for 1 h and then incubated with primary antibodies overnight. After rinsing, we incubated the immunocomplexes with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Promega, Madison, WI) and visualized the membranes with SuperSignal Chemiluminiscent substrate (Pierce, Rockford, IL) as previously described22, 59. We purchased primary antibodies from the following commercial sources: anti-phospho-Tak1 (1:1000; 4531S; Cell Signaling, Danvers, Massachusetts), anti-Tak1 (1:1000; MAB5307; R&D systems), anti-phospho-p65 (1:2000; 3033S; Cell Signaling), anti-p65 (1:2000; 06-418; Millipore, Billerica, Massachusetts), anti-phospho-Iκbα (1:1000; 9246; Cell Signaling), anti-Iκbα (1:1000; sc-371; Santa Cruz, Santa Cruz, California), anti-phospho-JNK (1:500; 9251; Cell Signaling), anti-JNK (1:1,000; 9258; Cell Signaling), anti-phospho-p38 (1:1000; 9215; Cell Signaling), anti-p38 (1:1000; 8680; Cell Signaling), anti-phospho-Erk (1:1000; 4284; Cell Signaling), anti-Erk (1:1000; 4696; Cell Signaling), anti-Traf6 (2µg for immunoprecipitation; 1:1000 for Western blot; sc-8409; Santa Cruz), anti-Nlk (2µg for immunoprecipitation; AB10206, Millipore), anti-Tab2 (1:1000; 3744; Cell Signaling), anti-Nfatc1 (1:1000; sc-7294; Santa Cruz), anti-HA (1:2000; H9658; Sigma-Aldrich), anti-Tbp (1:2000; T1827; Sigma-Aldrich) and anti-α-tubulin (1:10000; 75168; Sigma-Aldrich).

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Manufacturer protocol

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