Sc 56338

The western blotting assay was performed by well-established protocols as previously described (19 (link)). Primary antibodies used in this study were anti-ABL1 antibody (1:500, ab85947, Abcam, Cambridge, MA), anti-Bcl-2 antibody (1:300, BCL/10C4, Biolegend, San Diego, CA), Anti-Bcl-xl antibody (1:500, sc-136207, Santa Cruz Biotechnology, Dallas, TX), anti-Bax antibody (1:300, 2D2, Biolegend), anti-β-actin antibody (1:500, 2F1-1, Biolegend), anti-GAPDH antibody (1:500, FF26A/F9, Biolegend), anti-p27 antibody (1:300, sc-56338, Santa Cruz Biotechnology), anti-cyclin-D1 antibody (1:500, sc-8396, Santa Cruz Biotechnology), anti-IRS1 antibody (1:500, ab52167, Abcam), anti-AKT2 antibody (1:500, ab175354, Abcam), anti-PPP3CA antibody (1:10000, ab52761, Abcam), anti-TGFβ1 antibody (1:100, ab92486, Abcam), anti-MAP2K2 antibody (1:500, sc-81473, Santa Cruz Biotechnology), anti-PI3K-p11a antibody (1:1000, ab151549, Abcam). Secondary antibodies used were: anti-mouse IgG HRP-conjugated secondary antibody (1:5000, sc-516102, Santa Cruz Biotechnology), and anti-rabbit IgG HRP-conjugated secondary antibody (1:5000, sc-2357, Santa Cruz Biotechnology).

Cells cultured to 80%‐90% confluent were harvested and lysed in radio immunoprecipitation assay (RIPA; Invitrigon, USA) buffer added with 1% PMSF and 1% protease inhibitor cocktail (Boster, Wuhan, China) on ice. Protein concentrations were quantified using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by 10% SDS‐PAGE gel electrophoresis, then transferred to PVDF membranes. The membranes were blocked using 2% BSA in TBST (Beyotime, Shanghai, China) for 1.5 hour at room temperature. Membranes were incubated with primary antibodies against KIF20A (1:100,sc‐374508; Santa Cruz, CA, USA), Cyclin D1 (1:1000, 2922,CST, USA), Cyclin E1 (1:200,sc‐247; Santa Cruz, USA),p21 (1:1000, 2947,CST, USA),p27 (1:200,sc‐56338; Santa Cruz, USA) and actin (1:1000, 4970,CST, USA) overnight at 4°C. After being washed in TBST, membranes were incubated with 1:5000‐diluted a goat anti‐rabbit/mouse‐IgG secondary antibody conjugated with polymers of HRP (7074/7076, CST, USA) for 1 hour at room temperature. An ECL detection reagent (Beyotime, Shanghai, China) was used to visualize protein bands. Quantitative analyses of western blots were performed using Image J program (National Institutes of Health, Bethesda, MD, USA).