Western Blotting for Protein Analysis

The cells were harvested 24 h after treatment at the indicated doses and times, and the cell lysates were subjected to western blotting, performed as described previously (20 (link)). Briefly, the cells were collected and lysed using 10 mM Tris, 1 mM ethylenediaminetetraacetic acid, 10 mM KCl and 0.3% Triton X-100 (pH 7.9). The concentration of the protein samples was measured by the Bradford method. The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto Hybond-P polyvinylidene fluoride membranes (Amersham, Piscataway, NJ, USA). The membranes were subjected to western blot analysis with primary antibodies to caspase-8, -9 and -3, poly(ADP-ribose) polymerase (PARP), p110δ, Akt, phospho-Akt, p65, phospho-p65, cyclin-dependent kinase (CDK)4, CDK6, cyclin D1 and β-actin (Sigma-Aldrich). The secondary antibodies used in this study were provided by Multisciences Co., Ltd. (Hangzhou, China).