Glyceraldehyde 3 phosphate dehydrogenase

PCa cells were harvested at 72 hours after transfection, and total proteins were isolated by RIPA lysis buffer (Beyotime Biotech, Haimen, People’s Republic of China) supplemented with protease and phosphatase inhibitor cocktail (Hoffman-La Roche Ltd., Basel, Switzerland). Detection of total cellular protein concentration was conducted using a BCA protein assay kit (Beyotime). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Boster, Wuhan, People’s Republic of China), then transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The final blots were blocked in 5% nonfat dried milk for 1 hour at room temperature and then incubated overnight with the primary antibodies at 4°C. The primary antibodies were p53 (1:1,000), p21 (1:1,000) (both Cell Signaling Technology, Denver, MA, USA), Cyclin D1 (1:1,000), CDK4 (1:1,000), CDK6 (1:500) (all Affinity, Cincinnati, OH, USA), glyceraldehyde 3-phosphate dehydrogenase (1:1,000), and α-tubulin (1:500) (both Boster). The membranes were then incubated with the appropriate secondary antibodies for 1 hour at room temperature, before detection with an enhanced chemiluminescence assay kit (EMD Millipore).