Western blot analysis

Cells were harvested using the total protein extraction kit (KeyGEN BioTECH, Nanjing, China). Samples were incubated for 0.5 h on ice with agitation and then centrifuged at 14 000×g for 15 min. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Pierce). Protein samples were subjected to electrophoresis on SDS-polyacrylamide gradient gels, transferred to a PVDF membrane, and blocked in 5% non-fat milk in TBST (phosphorylated proteins were blocked in 5% BSA in TBST) for 2 h at room temperature. Blots were incubated with primary antibodies to the following proteins: ezrin (Abcam), cortactin (Abcam), E-cadherin (CST), α-SMA (Abcam), Slug (CST), Snail (Abcam), Twist (Abcam), Twist2 (Abcam), phosphorylated ezrin at Y-567 (CST), and GAPDH (Beyotime). GAPDH was used on the same membrane as a loading control. The signal was detected after incubation with anti-rabbit or anti-mouse IgG secondary antibody (Bioworld) coupled to peroxidase, using ECL (Millipore). Protein expression levels were evaluated by densitometric analysis.