Ripa ice cold lysis buffer

Cells were lysed with 40 μL RIPA ice-cold lysis buffer (Rockford, IL, USA) supplemented with protease inhibitor (Vazyme Biotech, Nanjing, China) for 30 min at indicated time points. The protein concentrations in the whole cell lysates were determined using the BCA (Rockford, IL, USA) method after centrifugation at 7000 g for 10 min at 4°C. The protein samples were separated depending on their molecular weight on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a transfer to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). 5% nonfat powdered milk in Tris-buffered saline was used to block the membranes, which were stained with primary antibodies overnight at 4°C. After that, the membranes were probed with a 1:10,000 dilution of a peroxidase-labeled secondary antibody. An ECL reagent (Rockford, IL, USA) was used to detect the antigen-antibody complexes. Primary antibodies against β-actin were obtained from SIGMA St. (Louis, MO, USA), p21Waf1/Cip1, and p27 from Cell Signaling Technologies (Beverly, MA, USA). The ratio of drug-treated samples to control samples was used to calculate the relative expressions, which were normalized by the quantified level of β-actin expression.