Western Blot Analysis of Protein Expression

Cells were lysed on ice with IP lysis buffer (Thermo Scientific, Waltham, MA) containing protease inhibitor cocktail set III (Millipore, Billerica, MA). Total proteins were separated by electrophoresis using Any kD precast polyacrylamide gel (Bio-Rad, Hercules, CA), and transferred onto PVDF membrane. After blocking with 5% skim milk (Thermo Scientific) in TBST buffer, membranes were incubated with the first antibody, respectively: anti-MELK monoclonal antibody (in-house, previously described [8 (link)]), anti-β-actin antibody, anti-p21 antibody, anti-FOXO1 antibody, anti-FOXO3 antibody, anti-pan-AKT antibody, anti-phospho-AKT (Thr308) antibody, anti-phospho-AKT (Ser473) antibody (Cell Signaling, Danvers, MA), and anti-p53 antibody (Sigma-Aldrich). β-actin was used as a loading control.