Immobilon polyvinylidene membranes

Proteins in cell lysates or concentrated supernatant media (CM) (concentrated by Ultracel-3K centrifugal filter units; EMD Millipore, Billerica, MA, USA) were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis following standard protocol and transferred to Immobilon polyvinylidene membranes (EMD Millipore). Membranes were blocked with 5% milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h followed by incubation with primary antibody (diluted in blocking buffer or 2% bovine serum albumin in TBST) overnight. Membranes were then washed three times in TBST and re-incubated with horseradish peroxidase-labelled secondary antibodies for 1 h, washed three times in TBST, and exposed to autoradiography films. Signals were detected by chemiluminescence (Thermo Fisher Scientific). Antibodies for N-cadherin, E-cadherin, pcMET Y1234/Y1235, pEGFR Y1068, pFAK Y925, pSRC Y416, vimentin, Snail, and Twist were from Cell Signaling Technology (Danvers, MA, USA). The antibody against pVEGFR1 (Y1213) was purchased from R&D Systems Inc.. Antibodies for VEGF, vinculin, α-tubulin, Zeb1, and actin were from Santa Cruz Biotechnology (Dallas, TX, USA). All antibodies were used according to manufacturers’ specifications. All cell lysates were prepared in radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors.