Western Blot Analysis of Protein Extracts

Cells were washed twice with PBS and lysed in radioimmunoprecipitation buffer composed of 20 mM Tris-HCl (pH 7.4), 0.1% sodium dodecyl sulphate (SDS), 1% Triton X-100, and 1% Na deoxycholate (all from Wako). Protein lysates (20 μg) were resolved by 10% Tris-glycine SDS polyacrylamide gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA) electrophoresis using an Any kD precast gel (Bio Rad Laboratories) and transferred to Immobilon polyvinylidene fluoride membranes (Millipore, Marlborough, MA, USA), which were blocked with Tris-buffered saline containing 5% dried milk (Megmilk Snow Brand Co., Ltd., Hokkaido, Japan). Western-blotted protein extracts from KhES-1 cells were probed with antibodies against KSP (originally produced by hybridomas13 (link)18 (link) or #ab116368; Abcam, Cambridge, MA) and β-actin (#A1978; Sigma-Aldrich) (loading control). The membranes were then incubated with alkaline phosphatase-conjugated anti-mouse or anti-rabbit secondary antibody (Promega, Madison, WI, USA), and horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Signals were detected using an alkaline phosphatase (Promega) or ECL detection system (Amersham).