Protein Extraction and Western Blotting Analysis

The extraction of total proteins from HCCLM3 cells was carried out by the use of an ice-cold RIPA buffer lysis (Beyotime Biotechnology, Shanghai, China). The concentration of the extracted proteins was measured with BCA protein assay kit (Sigma-Aldrich, St. Louis, MO, USA). 20 µg of protein samples were separated with the application of SDS-PAGE, followed by a transferring performed on PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies against DEPDC1B (Thermo Fisher Scientific, PA5-113,277, 1:500), cyclin-dependent kinase (CDK)1 (Abcam, ab201008, 1:1,000), CyclinD1 (Abcam, ab16663, 1:200), KIF23 (Thermo Fisher Scientific, A300-010A, 1:2,000), p53 (Abcam, ab32389, 1:10,000), p21 (Abcam, ab109520, 1:10,000) and GAPDH (Abcam, ab181602, 1:10,000) as the reference. Then the blots were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (Thermo Fisher Scientific, G-21234, 1:100,000). An enhanced chemiluminescence (ECL) detection kit (Cytiva, Shanghai, China) was adopted to develop these images of the protein bands. Finally, the quantification of bands was conducted with the help of Image J software (National Institutes of Health, Bethesda, MA, USA) [16 (link)].