Ecl reaction reagents

Cells were lysed on ice for 20 min using RIPA lysis buffer (Beyotime) with protease inhibitor cocktail (Sangon Biotech, China) and the protein lysates were collected. The protein concentration of each sample was determined using a Pierce BCA protein detection kit (Thermo Fisher Scientific). Protein lysates were boiled in 5× protein-loading buffer (Beyotime) at 95 °C for 10 min for denaturing, and then separated by 4–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands were transferred to a polyvinylidene difluoride membrane (Millipore, USA), blocked with 5% skim milk for 1 h, and the membrane was incubated with primary antibodies at 4 °C overnight. The membrane was then washed three times with Tris-buffered saline with Tween 20 (TBST), incubated with horseradish peroxidase-labeled secondary antibodies (Beyotime) at room temperature for 2 h, and washed three times with TBST. The protein bands were then visualized with ECL reaction reagents (Beyotime) using a Western Lightning Gel Imaging System (Tanon 4600; Tianneng Technology Corporation, Shanghai, China). Densitometric analysis of individual blot signals was carried out based on three independent western blot experiments using Image J software.