Western Blot Analysis of Apoptosis and Cell Cycle Proteins

Blotting was performed as described earlier [34 (link)]. In brief, the cells were harvested and lysed in RIPA buffer (Sigma-Aldrich). Cell lysates stored at -80°C until analysis. An amount of 20 μg proteins per sample were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane (Whatman Westran S). The membranes were blocked for 1 h in Western Blocker Solution (Sigma-Aldrich). Immunoblots were probed with rabbit anti-human caspase-3, 7, 9 and PARP monoclonal antibody (1:1000; Cell Signaling Technologies, USA) Whereas for CDKs studies CDK-2, 6, 7, 9 and CDC-2 monoclonal antibody (1:1000; Cell Signaling Technologies, USA) were used. Blots were allowed to incubate for 3 h with primary antibodies followed by horseradish peroxidase-linked anti-rabbit and anti-mouse IgG whole antibody (1:5,000; Cell Signaling Technologies, USA) for 1 h. All washing steps were performed using 0.05% PBST and all incubations were performed at room temperature. After incubation, the protein bands were visualized using ECL western blotting substrate (Thermo Scientific, Rockford, USA). All membranes were stripped and probed with anti β-tubulin antibody (Santacruz, USA) to use as loading control.