Anti cdh2 antibody

To detect protein, the cytoplasmic and nuclear protein extracts were prepared from the cells. The protein concentration of each sample was determined using a Nanodrop TM spectrophotometer (Thermo Fisher Scienti c). Protein (100 µg) from each sample were subjected to SDS-PAGE (4% stacking and 10% separating gels) and then transferred overnight onto polyvinylidene uoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight with the following antibodies: poly clonal anti-human TJP1 antibody (1:200, Abgent, San Diego, CA, USA); anti-CDH2 antibody (1:1,000, Abcam, Cambridge, UK); anti-aVIM antibody (1:1,000, Cell Signaling Technology, Danvers, MA, USA); anti-EPS15 antibody (1:500, Abgent); anti-GAPDH antibody (1:1,000, Santa Cruz Biotechnology, Dallas, TX, USA); and anti-Flag antibody (1:1,000, Cell Signaling Technology). Next, the blots were incubated with peroxidaseconjugated goat anti-rabbit antibody or goat anti-mouse antibody (1:4,000, Millipore) for 1 h. Finally, the membranes were subjected to immunoblotting analysis using an ECL immunoblotting kit according to the manufacturer's recommendations (Beyotime Institute of Biotechnology, Shanghai, China).