Western blot analysis of apoptotic signaling proteins

The AM cells from the 5 experimental groups were incubated in protein lysis buffer (BeyotimeInstitute of Biotechnology) for 30 min at 4°C with intermittent vigorous mixing. After centrifugation at 1000 rpm for 15 min at 4°C, the supernatant was stored at –80°C. The protein amount in the lysate was quantified using the Bradford assay kit (BioRad, Hercules, CA, USA). Then, the protein samples were separated on 10% SDS PAGE and transferred onto PVDF membrane (1.5 h). The initial voltage was 60 V, and elevated at 120 V when the front edge of the bromophenol blue into the seperation gel. the After blocking, the membrane was incubated with primary antibody (Anti-human Bcl-2, Caspase-3 and Caspase-8 and anti-human β-actin, dilution ratio of 1:2000, provided by the Santa cruz co., Ltd, CA, USA) for 1hfollowed by incubation with 1:2000dilutedgoat anti-rabbit secondary antibody for 45 min at 37°C. The blot was developed with ECL method (ECL reaction mixture, Santa cruz co., Ltd, CA, USA) and the protein bands were quantified by the image analysis software IPP 6.0.