X omat s films

Cells were washed once with ice-cold phosphate-buffered saline (PBS) and lysed with 0.1–0.2 ml RIPA lysis buffer. The protein concentration was determined by Coomassie protein assay (Pierce, Rockford, IL, USA). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, Hercules, CA, USA), and transferred onto a nitrocellulose membrane (Amersham, Chalfont, UK). The membranes were incubated with primary antibodies (aPKCι, Santa Cruz Biotechnology, Santa Cruz, CA, USA; E-cadherin, Cell Signaling Technology, Danvers, MA, USA; Par6, Santa Cruz Biotechnology; β-catenin, Cell Signaling Technology; Vimentin, Cell Signaling Technology; N-cadherin, R&D Systems, Minneapolis, MN, USA; v-Ha-Ras, Santa Cruz Biotechnology; Caspase 3, Santa Cruz Biotechnology) at 4°C overnight. Peroxidase-conjugated secondary antibodies were applied for 60 min at room temperature at a dilution of 1 : 2000. β-Actin (Santa Cruz Biotechnology) was used as the internal control. Peroxidase activity was detected by using chemiluminescence method (Amersham) and visualized on X-Omat S films (Amersham). To quantify the relative levels of protein expression, the intensity of the specific bands was estimated by the ImageJ2X analysis software package (National Institute of Mental Health, Bethesda, MD, USA).