Ab76059

Protein extraction was performed, and proteins expression was evaluated using a BCA Protein Assay Kit (Beyotime, China). The samples were then separated by 8%–10% SDS‐PAGE and transferred onto PVDF membranes (Bio‐Rad, USA). After blocking with 5% defatted dry milk, the samples were incubated overnight at 4°C with primary antibodies against BMPR1A (ab264043, 1/500, Abcam, USA), E‐cadherin (ab269767, 1 μg/mL, Abcam, USA), N‐cadherin (ab76059, 1/1000, Abcam, USA), vimentin (ab8069, 1 μg/mL, Abcam, USA), fibronectin (ab268021, 1/1000, Abcam, USA), and snail (ab63568, 1/500, Abcam, USA). Subsequently, the samples were then incubated with goat anti‐rabbit IgG H&L (HRP) preadsorbed (ab97080, 1/10000, Abcam, USA) or rabbit anti‐mouse IgG H&L (HRP) (ab6728, 1/2000, Abcam, USA) at 37°C for 1 h. The brands were visualized using the SuperSignal West Pico Chemiluminescence system (Pierce, Inc. USA) and analyzed with ImageJ software (NIH).

Total protein sample was extracted from cell lines with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Proteins of equal amounts (30 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies against PCNA (1:1000, ab18197, Abcam), CDK4 (1:1000, ab226474, Abcam), E-cadherin (1:1000, ab219332, Abcam), N-cadherin (1:1000, ab76059, Abcam), and GAPDH (1:5,000; ab8245; Abcam) overnight at 4 °C. After an incubation with horseradish-peroxidase-conjugated secondary antibody (1:5000, SC-2005, Santa Cruz, Inc.) for 2 h at room temperature, the protein bands were visualized with the enhanced chemiluminescence (ECL) Plus kit (Beyotime Institute of Biotechnology).