Total proteins were lysed by RIPA lysis buffer (Beyotime), followed by measurement with BCA Protein Assay Kit (Beyotime). After separation with a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system, the protein sample (40 μg) was further transferred to a nitrocellulose membrane (Millipore), followed by treatment with TBST containing 5% non-fat milk. After 2 h of incubation, the membranes were hybridized with primary antibodies: TRAF6 (1:1000; ab33915, Abcam, Cambridge, UK) and β-actin (1:5000; ab8227, Abcam) at 4°C. The next day, the membranes were probed with secondary antibody at room temperature for 1 h. With the help of an ECL detection kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the bands were detected, followed by analysis with Quantity One software (Bio-Rad, Hercules, CA, USA).

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