Cells were plated in six-well plates at a density of 106 cells/mL to extract total proteins using the Q-proteome Mammalian Protein kit. Proteins were quantified using Lowry assay. Western blot analysis was done to measure the protein expression of p53, p21, Bax, Bcl2, cytochrome c, PARP, and caspase-3 and -9.
Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins were separated on 10% gels and transferred to polyvinylidene difluoride membranes at 0.25 mA for 75 min. The membranes were then blocked with 5% skimmed dry milk in PBS containing 0.05% Tween 20 for a night at 4°C. Membranes were then incubated with primary antibody using anti-β-actin, anti-p53, anti-p21, anti-Bax, anti-bcl2, anti-caspase-3 and anti-caspase-9 antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) at a concentration of 1:500. After washing the membranes for 1 h using 1× PBS with 0.5% Tween 20, they were incubated with secondary antibody (obtained from Promega, Madison, WI, USA) at a concentration of 1:2500 for 1 h at room temperature. The membranes were then washed and the development was done using Western blotting chemiluminescent reagent enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA) He and figures were taken using ChemiDoc XRS+ machine (Bio-Rad, Hercules, CA, USA).
Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins were separated on 10% gels and transferred to polyvinylidene difluoride membranes at 0.25 mA for 75 min. The membranes were then blocked with 5% skimmed dry milk in PBS containing 0.05% Tween 20 for a night at 4°C. Membranes were then incubated with primary antibody using anti-β-actin, anti-p53, anti-p21, anti-Bax, anti-bcl2, anti-caspase-3 and anti-caspase-9 antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) at a concentration of 1:500. After washing the membranes for 1 h using 1× PBS with 0.5% Tween 20, they were incubated with secondary antibody (obtained from Promega, Madison, WI, USA) at a concentration of 1:2500 for 1 h at room temperature. The membranes were then washed and the development was done using Western blotting chemiluminescent reagent enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA) He and figures were taken using ChemiDoc XRS+ machine (Bio-Rad, Hercules, CA, USA).