Peroxidase conjugated goat anti rabbit antibody or goatanti mouse antibody

For the detection of protein, cytoplasm and nuclear protein extracts were prepared from cells. The protein concentration of each sample was determined using a NanodropTM spectrophotometer (Thermo Scienti c). Protein (100ug) from each sample was examined bySDS-PAGE(4% stacking and 10% separating gels) and then transferred overnight on to PVDF membranes(Millipore). The membranes were immunoblotted with the following: poly clonal anti-human TJP1 antibody (1:200, Abgent); CDH2 antibody (1:1000, Abcam); VIM antibody (1:1000, Cell Signaling Technology); EPS15 antibody (1:500, Abgent); GAPDH antibody (1:1000, Santa Cruz Biotechnology); Flag antibody (1:1000, Cell Signaling Technology) overnight. The blots were then incubated with peroxidase-conjugated goat anti-rabbit antibody or goatanti-mouse antibody (1:4000, Millipore) for 1 h. The PVDF membranes were subsequently subjected to immunoblotting analysis using an ECL immunoblotting kit according to the manufacturer's recommended protocol (Beyotime Institute of Biotechnology, China).

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).