Protein extraction reagent ripa buffer

Total protein from cells was isolated using protein extraction reagent RIPA buffer (Beyotime Institute of Biotechnology). The protein concentration was measured using the BCA protein assay kit (Beyotime Institute of Biotechnology). A total of 40 µg protein was added to 10% SDS-PAGE and separated proteins were transferred onto polyvinylidene fluoride membranes, which were then blocked with 5% non-fat milk in TBS at room temperature for 1 h. Subsequently, these membranes were washed with TBS-Tween-20 (1X TBS, 0.1% Tween-20 and 5% nonfat milk powder; Wuhan Boster Biological Technology, Ltd.) three times and incubated with antibodies targeting caspase-3 (1:500; cat. no. ab49822; Abcam), SOX6 (1:1,000; cat. no. ab64946; Abcam) or β-actin (1:5,000; cat. no. ab179467; Abcam) overnight at 4˚C. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody targeting anti-rabbit IgG (1:5,000; cat. no. ab6721; Abcam) for 1 h. The protein signals were determined using an ECL detection kit (Thermo Fisher Scientific, Inc.).