Anti caspase 3 casp3

After washing in cold PBS twice, GCs were treated with RIPA lysis buffer (Beyotime, 243 Shanghai, China) containing 1.5% proteasome inhibitor (PMSF, Solarbio, Beijing, China) for 20 min to isolate total protein. The concentration of each protein sample was quantified by using a BCA Kit (Biosharp, Beijing, China). A total of 15 μg of protein from each sample was loaded on 15% polyacrylamide gel and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GenScript, Nanjing, China). After separation by electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% nonfat milk for 1 h and then incubated with primary antibodies for 8 h at 4 °C. The bound primary antibody was visualized with secondary antibody using an ECL detection reagent (Advansta, CA, USA) with a LAS2000 imaging system (GE Healthcare, Chicago, IL, USA). The β-tubulin protein level was used as the internal control. The primary antibodies used in the study were anti-Caspase 3 (CASP3) (diluted at 1:1,000; Proteintech, Wuhan, China), anti-FoxO1 (diluted at 1:1,000; CST, BMA, USA), and anti-β-tubulin (diluted at 1:2,000; Proteintech, Wuhan, China).