Western blot analysis

Phosphate-buffered saline was used to wash the cells harvested or the tissue samples, followed by lysing them in radioimmunoprecipitation buffer containing phosphatase/protease inhibitor cocktail purchased from Thermo Scientific (Tokyo, Japan). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with each protein sample, then we transferred the protein to a nitrocellulose membrane and incubated them with anti-CD44 and anti-β-actin primary antibodies (SCBT, Santa Cruz, CA). After incubating with secondary antibodies (SCBT, Santa Cruz, CA), the Enhanced Chemiluminescence Detection System, purchased from GE Healthcare, was used to detect signals (Little Chalfont, UK).