Anti snail

Cell and tissue samples were lysed in radioimmune precipitation assay buffer (150 mM NaCl, 0.5% sodium deoxycholate, 50 mM Tris-HCl, pH 8, 0.1% SDS, 0.1% Nonidet™ P -40) supplemented with protease inhibitors (Roche AG, Switzerland). Proteins were separated on SDS/PAGE gels, transferred to nitrocellulose membranes, and blocked with 5% nonfat milk in PBS containing 0.1% Tween® 20. The membranes were then probed overnight at 4°C with the relevant primary antibodies: anti-E-cadherin (Cell Signaling Technology, MA, USA), anti-vimentin (Cell Signaling Technology), anti-SNAIL (Thermo Fisher Scientific, MA, USA), anti-ZEB1 (Cell Signaling Technology), anti-OCT3/4 (Cell Signaling Technology), anti-NANOG (Cell Signaling Technology), and anti-GAPDH (Cell Signaling Technology). After extensive washing, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies (Cell Signaling Technology). Blots were detected using an enhanced chemiluminescence detection kit (Amersham BioSciences, UK) and revealed using the ChemiDoc^TM XRS System (Bio-Rad Laboratories, CA, USA).

Western blot analysis was performed according to our standard procedures as described in earlier studies [16 (link), 17 (link)]. Briefly, equal amounts of cellular protein were separated by SDS-PAGE and transferred onto polyvinylidenefluoride (PVDF) membranes (Bio-Rad, Hercules, CA). The membrane was blocked with 5% non-fat milk for 1 hour, and subsequently incubated with primary and secondary antibodies. Immunoreactive bands were visualized with a chemiluminescence detection system (Thermo Scientific Pierce, Rockford, IL). All antibodies used in these studies were purchased from Cell Signaling Technology (Danvers, MA) with the following exceptions: anti-OLA1, Abcam (Cambridge, MA); anti-β-actin, Sigma-Aldrich; anti-Snail, Thermo Scientific; and anti-rabbit (or mouse) IgG, peroxidase-linked secondary antibody, GE Healthcare (Pittsburgh, PA).