Ripa lysis buffer

Total protein was extracted with RIPA lysis buffer (ABclonal, Wuhan, China), and quantified by bicinchoninic acid (BCA) assay. After sample preparation, protein samples (20 μg) were separated by electrophoresis on a 10% SDS-PAGE gel and transferred to a PVDF membrane. After blocking with 5% nonfat milk in TBST for 2 h at room temperature, the membrane was incubated overnight at 4 C with the following primary antibodies: anti-USP25 (diluted 1:1,000, A7975, ABclonal, Wuhan, China), anti-TRAF6 (diluted 1:500, D21G3, Cell Signaling Technology, Boston, United States) and anti-GAPDH (diluted 1:10,000, ab181602, Abcam, Cambridge, United Kingdom). The membrane was then incubated with secondary antibody (anti-rabbit, 1:10,000, BS13278, Bioworld, Minnesota, United States) for 2 h at room temperature. After washing three times in TBST, protein bands were visualized with enhanced chemiluminescence (ECL) reagent (ABclonal, Wuhan, China). The gray values of the protein bands were quantified with ImageJ software (ImageJ 1.53, NIH, United States). Samples with poor expression of GAPDH were excluded, and the results were statistically evaluated by Student’s t test. Differences with a p value <0.05 were considered statistically significant.

RIPA lysis buffer (ABclonal Technology Co., Wuhan, China) was used to lyse nuclear and cytoplasmic proteins. Protein lysates were boiled for 10 min at 100°C in SDS sample buffer and transferred to PVDF membranes after separation on SDS-polyacrylamide gels. Next, 5% skim milk was used to block the mixture. After blocking, the membranes were incubated overnight at 4°C with primary antibodies, followed by incubation with secondary antibodies for 1 h at room temperature. Blots were analyzed using a chemiluminescence imaging system (Tanon, Shanghai, China).