RIPA lysis (ThermoFisher, USA) was used for extracting total cell protein. Protein concentration was tested by BCA assay (ThermoFisher, USA). Equal amounts of protein were separated by SDS-PAGE gel as regular protocol. And the protein was transferred from gel to a PVDF membrane (Millipore). Then the PVDF membrane was blocked in 5% BSA-TBST (Sigma, USA) for 2 h at room temperature, followed by primary antibody at 4-degree overnight. The membrane was washed 10 min 3 times with TBST, followed by secondary antibody at room temperature for 1 h. After another washing cycle, the membrane was visualized by ultra-sensitive ECL kit. Results was calculated by Target protein/ β-actin based on band intensity tested by ImageJ.
The related antibodies used were as follows:
E-cadherin (CST, Boston, USA,14472), N-cadherin (CST, Boston, USA, 13116), Vimentin (CST, Boston, USA, 5741), Snail (CST, Boston, USA), MMP9(CST, Boston, USA), smad2/3((abcam, USA, ab202445), p-smad2/3 (CST, Boston, USA),BMP4 (abcam, USA, ab124715),P-SMAD1/5/8(CST, Boston, USA, 13820), Anti-SMAD1/5/8 antibody (abcam, USA,ab80255), and TGF-BETA1 (abcam, USA, ab179695), SMAD6(SANTA CRUZ, USA, sc-25,321).
The related antibodies used were as follows:
E-cadherin (CST, Boston, USA,14472), N-cadherin (CST, Boston, USA, 13116), Vimentin (CST, Boston, USA, 5741), Snail (CST, Boston, USA), MMP9(CST, Boston, USA), smad2/3((abcam, USA, ab202445), p-smad2/3 (CST, Boston, USA),BMP4 (abcam, USA, ab124715),P-SMAD1/5/8(CST, Boston, USA, 13820), Anti-SMAD1/5/8 antibody (abcam, USA,ab80255), and TGF-BETA1 (abcam, USA, ab179695), SMAD6(SANTA CRUZ, USA, sc-25,321).