Western Blot Analysis of Protein Expression

Cells were collected and then lysed in a RIPA lysis buffer with phenylmethanesulfonyl fluoride (PMSF) as previously described [31 (link)]. Protein concentration was detected with a BCA protein assay kit. The protein was separated on 8–12% SDS-PAGE Gels, and then transferred to a polyvinylidene fluoride (PVDF) membrane. After being blocked with 5% BSA at room temperature for 2 h, the PVDF membrane was incubated with a diluted primary antibody overnight at 4 °C. The next day, the PVDF membranes were incubated with horseradish peroxidase-conjugated secondary antibody (HRP-conjugated secondary antibodies) at room temperature for 2 h. Finally, the results were analyzed with the Super ECL prime (US Everbright®Inc., Suzhou, China) and western blotting detection system (Qin Xiang, Shanghai, China).