Western blot

HCEC-21T and FECD-54F CECs were plated in 100 mm cell culture plate pre-coated with undiluted fibronectin coating mix and allowed to grow for 48–72 h until cells reached 90–100% confluency at time of protein lysate collection. Whole cell lysates were prepared in a RIPA Buffer (50 mM Tris, pH 8, 150 mM NaCl, 5 mM EDTA, pH 8, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS) containing 1 × protease inhibitor cocktail (Millipore Sigma, Oakville, Ontario) and 1 mM PMSF. Lysates were separated on an SDS-PAGE gel and transferred onto Amersham hybond ECL nitrocellulose membrane (Millipore Sigma, Oakville, Ontario). Membranes were blocked with 5% skim milk and subsequently incubated with the indicated antibodies at 4 °C overnight. Appropriate HRP-conjugated secondary antibodies were incubated for one hour at room temperature. Signals were detected using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific, Waltham, MA) or Pierce SuperSignal West Pico Plus Chemiluminescent substrate (ThermoFisher Scientific, Waltham, MA). The primary and secondary antibodies and the concentrations used were: rabbit anti-TCF-4 (1:1000, Proteintech, Rosemont, IL), rabbit anti-Flag (1:1000, Cell Signaling, Danvers, MA), mouse anti-Fibronectin (1:1000, Santa Cruz, Dallas, TX), rabbit anti-ZEB1 (1:1000, Cell Signaling, Danvers, MA), mouse anti-N-cadherin (1:1000, Santa Cruz, Dallas, TX), rabbit anti-Snail (1:1000, Cell Signaling, Danvers, MA), mouse anti-Vimentin (1:1000, Santa Cruz, Dallas, TX), mouse anti-TUBB4A (1:1000, Abcam, Waltham, MA), rabbit anti-Vinculin (1:4000, Abcam, Waltham, MA), mouse anti-Actin (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-GAPDH (1:5000, Cell Signaling, Danvers, MA), anti-mouse (1:20,000, ThermoFisher Scientific, Waltham, MA), anti-rabbit (1:20,000, ThermoFisher Scientific, Waltham, MA).