Protein extraction and western blot

Cells were lysed for 30 min on ice in RIPA lysis buffer (10 mM Tris [pH 8.0], 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.5% deoxycholate), supplemented with a protease inhibitor PMSF, and they were centrifuged at 14,000 × g for 30 min at 4°C and the supernatants collected. SDS-polyacrylamide gel electrophoresis and western blotting were performed in accordance with standard protocols. Antibodies to mouse monoclonal antibodies RASSF1A and CDK4 (Abcam, Cambridge, UK) and rabbit polyclonal antibody β-actin (Boshide, Wuhan, China) were all diluted at 1:1000. Secondary antibodies were all diluted at 1:4000. Image J software was used to quantify and analyze the density of the protein bands.