2.8. Western blot analysis of apoptotic markers expression

The Western blot was carried out by the method of Towbin et al. [26]. Immunoblot analysis was carried out for p53, Bax, Bcl-2, Cytochrome c, Caspase-9 and Caspase 3 protein expressions. Cultured cells were washed with PBS solution and detached with trypsin. Cell suspensions were centrifuged at 1000 rpm for 10 min and the pellets were lysed with a protease inhibitor cocktail present in chilled lysis RIPA buffer for 30 min. Then by centrifugation for 10 min at 4 °C with 14,000 rpm, the lysate was cleared and the supernatant was taken for Western blotting analysis. The protein concentration of obtained supernatant was determined by Nanodrop (Thermo Scientific, USA). Cell extracts containing 50 μg of proteins were fractionated on SDS-PAGE gel electrophoresis (12%), and then the separated molecules were blotted onto a polyvinylidene fluoride (PVDF) membrane. After blocking, the respective primary antibody was added and was allowed to bind to the protein. It was followed by washing (which removes nonspecifically bound antibody) and secondary antibody was added, to detect the primary antibody. TBST was used for washing the membranes thrice, with 10 min interval, and the bands were analyzed using western blotting chemiluminescence substrate (LI-COR, USA). The density of band was analyzed by Image studio software (LI-COR, USA).