0.22 mm nitrocellulose membranes

The cells were lysed using mammalian protein extraction reagent RIPA (Beyotime, Haimen, China) supplemented with protease inhibitors cocktail (Roche) and PMSF (Roche). Fifty micrograms of the protein extractions were separated by 10% SDS-PAGE transferred to 0.22 mm nitrocellulose membranes (Sigma-Aldrich, St. Louis, MO, USA) and incubated with specific antibodies.The autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad, Hercules, CA, USA). Anti-p53 was from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-cleaved caspase3, anti-Bax, anti-Bad and anti-Bcl-2 were from Cell Signaling Technology (Boston, MA, USA). Anti-NF-YA was from Abcam (Hong Kong, China). Results were normalized to the expression of GAPDH (Rabbit anti-GAPDH).

The cells were lysed using the mammalian protein extraction reagent, RIPA (Beyotime, Shanghai, China), supplemented with a protease inhibitor cocktail (Roche) and PMSF (Roche). Protein were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to 0.22 mm nitrocellulose membranes (Sigma) and incubated with specific primary antihuman antibodies. The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG. An ECL chromogenic substrate was used to visualize the bands and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad,Hercules, CA, USA). A GAPDH (#2118, CST, USA) antibody was used as a control. The anti-CDK4 (#12790, CST, USA), p21 (#3698, CST, USA), cleaved caspase-3 (#9664, CST, USA) and cleaved caspase-9 (#9509, CST, USA) (all 1:1000) antibodies were purchased from Cell Signaling Technology, Inc. (CST). The anti-KLF2 (SAB1101046, Sigma) antibody was purchased from Sigma.

12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate protein lysates. Then, the protein lysates were transferred to 0.22 mm nitrocellulose membranes (Sigma) with particular antibodies incubation. Antibodies aganist CDK2, CDK4, cleaved caspase-3, and cleaved caspase-9 were supplied by Cell Signaling Technology, Inc. (CST). Antibodies aganinst P15 and KLF2 were supplied by Sigma.