Anti topk antibody

After washing with phosphate‐buffered saline (PBS) twice, cultured cells were harvested and kept at −80°C until use. For western blot analysis, total proteins were extracted from cells using the IP lysis buffer (Thermo Scientific, Waltham, MA, USA) supplemented with the protease inhibitor cocktail III (EMD Millipore, Billerica, MA, USA). Then, the proteins were separated by electrophoresis using 4–20% SDS‐PAGE gel (Bio‐Rad) and transferred onto PVDF membranes. After blocking with skim milk (EMD Millipore) overnight, the membranes were incubated with the first antibody, respectively: anti‐TOPK antibody (BD Biosciences, Franklin Lakes, NJ, USA), anti‐FOXM1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti‐phospho‐FOXM1 antibody (Cell Signaling Technology, Danvers, MA, USA). We generated a mouse anti‐MELK (maternal embryonic leucine‐zipper kinase) monoclonal antibody using partial recombinant MELK protein of 264–601 amino acids.7 An anti‐β‐actin antibody (Sigma‐Aldrich) was used as a loading control.