Apoptosis and autophagy-related protein evaluation by Western blot analysis

For tissue protein extraction, tissues were lysed in radio-immunoprecipitation assay buffer; then, the lysates were centrifuged for 10 minutes at 13,000× g, and the supernatant was retrieved. For cell protein extraction, the cells from each group were harvested and lysed with radio-immunoprecipitation assay (120 µL) buffer on ice 24 hours after drug treatment. Immunoblotting assays were conducted as previously described.33 In brief, a BCA Assay Kit (Beyotime Biotech, Haimen, People’s Republic of China) was used to determine the protein concentrations of the lysates. Afterward, samples were resolved in sample loading buffer (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), heated up to 100°C for 5 minutes, and cooled on ice for 5 minutes. Appropriate levels of lysate (30 µg protein/sample) were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then transferred to polyvinylidene difluoride (PVDF) membranes. Nonfat milk (5%) was used to block the PVDF membranes for 1 hour at room temperature. Then, the membranes were incubated at 4°C overnight with primary antibodies Beclin-1, LC3, p62, Bax, Bcl-2, and GAPDH that were diluted according to the manufacturer’s recommendations. Afterward, the PVDF membranes were completely washed using 0.1% (V/V) Tris-buffered saline with Tween-20. The membranes were later incubated with the corresponding secondary antibodies (horseradish peroxidase-conjugated; Santa Cruz Biotechnology, Dallas, TX, USA). According to the manufacturer’s protocol, we used an electrogenerated chemiluminescence system to visualize the corresponding secondary antibody. ImageJ 1.33 software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the protein bands.