Mouse anti beta actin

Proteins were extracted using RIPA buffer (Solarbio, Beijing, China, R0020) according to the manufacturer’s protocol. The concentration of protein was measured by BCA kit (Takara, Japan, AI90451A). Thirty micrograms of total proteins from each sample were separated by polyacrylamide gel electrophoresis and transferred onto poly-vinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The blot-transferred membranes were blocked with 5% fat-free dry milk for 2 h and incubated with primary antibodies: mouse anti-PCNA (1:500; Santa Cruz Biotechnology, PC10), mouse anti-CDK1 (1:500; Santa Cruz Biotechnology, AN21.2), mouse anti-CDK2 (1:500; Santa Cruz Biotechnology, D-12), and mouse anti beta-ACTIN (1:2000; CWBIO, Beijing, China, CW0096) on a shaker at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)–conjugated anti-mouse IgG (1:2000; Millipore, AP192P). Protein bands were visualized and captured by a Bio-Rad Chemidoc XRS using a Western Bright ECL Kit (Bio-Rad, Berkeley, CA, USA). The protein bands were hand-drawn for 3 times and calculated the average intensity by Image-Pro Plus. The ratio of the target protein to the corresponding intensity value of β-actin was obtained for analysis. Experiments were repeated three times.