Amplified opti 4cn

Cell lysates were prepared in Tris-HCl 50 mM pH 7.6, 150 mM NaCl, 1% Triton X-100, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% sodium dodecyl sulphate (SDS), proteases inhibitors, and 1 mM sodium orthovanadate. Lysates were incubated on ice for 30 min and centrifuged at 14,000 g for 10 min at 4 °C to remove cell debris. Cell lysates (40 µg of total proteins) were diluted in SDS sample buffer, loaded on 10% SDS polyacrylamide gel, separated under reducing and denaturing conditions at 80 V according to Laemmli, and transferred at 90 V for 90 min to a nitrocellulose membrane in 0.025 M Tris, 192 mM glycine, and 20% methanol, pH 8.3. For E-cadherin evaluation, membranes were incubated for 1 h at room temperature with monoclonal antibodies to E-cadherin (1:2500, Becton Dickinson) and, after washing, in horseradish peroxidase (HRP)-conjugated rabbit anti-mouse serum (1:40,000 dilution, Sigma-Aldrich). To confirm equal loading, membranes were reprobed by monoclonal antibody to α-tubulin (1:2000 dilution, Sigma-Aldrich). Immunoreactive bands were revealed using the Amplified Opti-4CN (Bio Rad, Hercules, CA, USA).