Ab63568

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).

Equal amounts (70 μg) of protein lysate were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% (w/v) nonfat milk in Tris-buffered saline with 0.1% Tween (TBST) for 1 h and incubated with a rabbit monoclonal primary antibody against E-cadherin (ab40772; 1:1000; Abcam), N-cadherin (ab195186; 1:8000; Abcam), vimentin (ab45939; 1:1000; Abcam), Slug (ab63568; 1:800; Abcam), YAP1 (ab205270; 1:600; Abcam) and rabbit polyclonal anti-GAPDH (ab9485; 1:4000; Abcam) overnight at 4 °C. After a incubation with the corresponding horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Sigma, USA) at 37 °C for 1 h, the protein bands were visualized by the enhanced chemiluminescence (ECL) Plus kit (Beyotime, Shanghai, China). The blots were detected on Kodak film developer (Fujifilm, Japan).