Western Blot Analysis of EMT Markers

This method was performed as described previously^{17 (link)}. The cells grown on plates were trypsinized, and detached cells were collected by centrifugation. The cell pellet was lysed with cold lysis buffer supplemented with protease inhibitors. Protein samples (30 µg) from each cell lysate were equivalently loaded on a precast gel and used for electrophoresis. Gels were subsequently blotted onto nitrocellulose membranes (0.45 μM; Bio-Rad, Hercules, CA), followed by blocking nonspecific binding with a solution containing 1 × PBS, 0.1% Tween-20, and 5% nonfat dry milk powder at room temperature for 1 h. Membranes were incubated with the primary antibodies anti-E-cadherin, anti-Snail, anti-Vimentin, anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA) and anti-Sema4C (Abcam, Cambridge, MA, USA) at 4 °C overnight. After extensive washing with TBST, horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad) were incubated for 1 h at room temperature. Bands were detected with an enhanced chemiluminescence kit (Super Signal West Pico Substrate; Pierce, Rockford, IL, USA). Quantification of signal intensities was performed by densitometry on a Xerox scanner using NIH ImageJ software (ImageJ, Bethesda, MD).