Tgfβr1

Western blot analysis was performed as previously described 21 (link), tissues and cells were washed with PBS, and lysed with RIPA lysis buffer including protease lysis inhibitor (Roche USA) and phosphatase inhibitor (Roche USA). A BCA protein assay kit (Thermo Scientific, USA) was used to measure protein concentration. 30ug protein extract was separated with a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane (microwell). The membrane was blocked with TBST buffer containing 5% skim milk at 37 °C for 2 hours, incubated with primary antibody at 4 °C overnight, and incubated with peroxidase-conjugated secondary antibody for 2 hours. The signal was detected using ECL kit (Cell Signaling Technology, 12757) and photographed. Bands were quantified using gray-scale analysis software (Bio-Rad). GAPDH expression is used as a loading control to standardize the expression of other proteins. The main antibodies are as follows: anti-ADNP (1: 1000, Proteintech, USA), anti-GAPDH, N-Cadherin, Vimentin, Snail, E-Cadherin, β- Catenin, Claudin-1, TGF-β, TGF-βR1, Smad2/3, p-Smad2/3 (1: 1000, Cell Signaling Technology, USA). The secondary antibodies are as follows: HRP-goat anti-rabbit Ig G, HRP-goat anti-mouse Ig G (1: 5000, Cell Signaling Technology, USA).