Western Blot Analysis of P53 and β-actin

Western blot was performed strictly according to previously described procedures (Chen et al., 2022 (link)). Briefly, cells were washed with ice-cold PBS and soluble proteins were extracted with RIPA Lysis Buffer (Strong) (Beyotime Biotech, Shanghai, China) according to the manufacturer’s protocol. The protein concentration of each sample was determined by a BCA Kit (Beyotime Biotech, Shanghai, China). An equal amount of protein was separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore, Billerica, MA). The membrane was then blocked with 5% non-fat milk for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. Antibodies against P53 (Santa Cruz Biotechnology, Santa Cruz, USA) and β-actin (Proteintech, Wuhan, China) were used. Signals were visualized using infrared imaging systems (Odyssey CLX, LiCor Biosciences, Lincoln, NE).