Anti rabbit antibodies

Protein was obtained from the Cal27, SCC61 and SCC090 cells, and lysed with RIPA buffer (50 mm Tris-HCl pH 8.0, 150 mm NaCl, 1% IGE-PAL CA 630, 0.5% Na-DOC, and 0.1% SDS). Total protein concentrations were measured using Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc.). 10 µl Equal amount of protein was set on Mini-PROTEAN TGX gels (Bio-Rad Laboratories, Inc.). The following primary antibodies were added to nitrocellulose membranes with 5% non-fat dry milk in Tris-buffered saline and 1% Tween-20, and incubated at 4°C overnight: NOTCH4 (1:500, #2423, Cell Signaling Technology, Inc.), HES1 (1:1,000, #sc-25392, Santa Cruz Biotechnology, Inc.), HEY1 (1:400, #ab22614, Abcam), E-cadherin (1:10,000, #610181, BD Biosciences), fibronectin (1:3,000, #ab2413, Abcam), Vimentin (1:500, #V6630, Sigma-Aldrich; Merck KGaA), TWIST1 (1:1,000, #sc-15393, Santa Cruz Biotechnology, Inc.) and SOX2 (1:1,000, #2748, Cell Signaling Technology, Inc.). HRP-conjugated goat anti-mouse (#1010-05, 1:20,000 dilution; SouthernBiotech) or anti-rabbit antibodies (#4010-05, 1:20,000 dilution; SouthernBiotech) were used as secondary antibodies. These secondary antibodies were incubated at room tempera-ture for 1 h. Western blots were developed using Pierce ECL Western Blotting Substate (Thermo Fisher Scientific).