Western blot analysis

Cells were lysed on ice in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM Na₃VO4₂, 1 mM EDTA, and 1 mM EGTA). Total protein extracts were analysed by western blotting, as described previously [18]. Proteins (20 µg) were separated by SDS-PAGE gels (Invitrogen) and transferred to PVDF membranes. The membranes were blotted for 1 h with 5% milk. Membranes were incubated with primary antibodies (1 : 500 dilution) against E-cadherin, N-cadherin, or ERK-1 (Santa Cruz Biotechnology, Inc., USA) at 4°C overnight. After incubation with horseradish peroxidase-conjugated secondary antibody (1 : 1000 dilution) for 3 h at 37°C, signals were detected by ECL chemiluminescence for 5 min. The films were analysed by densitometry with image software.