Transfected cells were collected and lysed in lysate buffer (Beyotime, Beijing, China). Protein concentration was determined using bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA), and protein samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membrane (Whatman, Dassel, Germany). After blocking with 5% nonfat milk for 2 h, the membranes were incubated with the primary antibodies overnight, and horseradish peroxidase-conjugated secondary antibody was used for 2 h. Finally, protein bands were visualized using the enhanced chemiluminescence (ECL) reagent (GE Healthcare, Little Chalfont, UK), and optical density was calculated by ImageJ software version 1.49 (National Institutes of Health, Bethesda, MD, USA).
Antibodies used were as follows: actin (C-2) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p27 (ab54563), p21 (ab80633), Bcl-2 (ab692), Bax (ac32503), cleaved caspase 3 (ab32042), procaspase 3 (ab2171), and horseradish peroxidase-conjugated anti-mouse (ab6785) and anti-rabbit antibodies (ab6721) were purchased from Abcam (Cambridge, MA, USA).
Antibodies used were as follows: actin (C-2) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p27 (ab54563), p21 (ab80633), Bcl-2 (ab692), Bax (ac32503), cleaved caspase 3 (ab32042), procaspase 3 (ab2171), and horseradish peroxidase-conjugated anti-mouse (ab6785) and anti-rabbit antibodies (ab6721) were purchased from Abcam (Cambridge, MA, USA).