Western Blot Analysis of Apoptosis-Related Proteins

After incubation with control treatment, DFP, VC, and DFP and VC for 48 hours, approximately 2 × 10⁶ cells were harvested and lysed in 200 µL of RIPA (radioimmunoprecipitation assay) lysis buffer by “freeze-thaw” at −80°C to obtain total protein extracts. The protein concentration was determined using the BCA protein assay (Erwan Biotechnology Co, Shanghai, China). Equal amounts of cell lysate (20 µg) were first subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; Sigma Co, St Louis, MO, USA) and then transferred to a nitrocellulose membrane. The blot membrane was incubated for 90 minutes with the primary antibodies against Bcl-2, BAX, or poly(ADP-ribose) polymerase (PARP) (Immunoway Biotechnology Co, Plano, Texas, USA), followed by a 30-minute incubation with the secondary antibody conjugates. The specific immunoreactive protein bands were then detected by chemiluminescence using the manufacturer’s protocol (Sigma Co, St. Louis, MO, USA).