Western Blot Analysis of Cell Proteins

Western blot analysis was performed as previously described [36 (link), 37 (link)]. In brief, total protein was extracted from the cells using RIPA lysis buffer (CWBIO, Beijing, China) and quantified with a Protein BCA Assay Kit (Bio-Rad, Hercules, California, USA). The protein was then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA). The PVDF membrane was then blocked in 5% powdered milk at room temperature for 1 h followed by incubation with rabbit anti-CCND1, anti-CCND2, and anti-GAPDH antibodies (1:1000, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. After washing and incubation with a goat-anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP) (1:1000, Cell Signaling Technology), protein bands were detected by a chemiluminescent HRP substrate (Millipore) and imaged with an E-Gel Imager.