Western Blot

After 48 h of transfection, cells were washed three times with cold PBS. Then proteins were extracted from cells on ice by whole cell lysate, and the concentration was assayed using BCA kit (Thermo Fisher Scientific, Waltham, MA, United States). 30 μg of the total extraction was separated through polyacrylamide gel electrophoresis (PAGE), and transferred onto the PVDF membranes (Amersham, United States) that were sequentially blocked in 5% skim milk for 1 h. Afterward, the membrane was incubated with primary rabbit polyclonal antibodies overnight at 4°C, followed by horseradish peroxidase (HRP)-labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1:2000, Abcam, Cambridge, United Kingdom) at room temperature for 1 h. Primary antibodies contained CCNA2 (ab181591, 1:2000, Abcam, Cambridge, United Kingdom), p53 (ab32389, 1:1000, Abcam, Cambridge, United Kingdom) and GAPDH (ab9485, 1:2500, Abcam, Cambridge, United Kingdom). PBST (PBS containing 0.1% Tween-20) was utilized to wash the membranes three times following each step. Protein bands were visualized by chemiluminescence apparatus (GE, United States) and then photographed.