Gapdh g8140

Total proteins were collected with RIPA lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and protein concentrations were measured following the BCA method (Beyotime, Jiangsu, China), and 40ug protein were subjected to 4–20% SDS gel electrophoresis (Sigma) and were then transferred to PVDF membranes (Roche, Indianapolis, IN, USA). Then, 5% milk blocked membranes were incubated with TWIST1 (ab50581, Abcam, Cambridge, MA, USA), E-cadherin (ab1416, Abcam) or Vimentin (ab92547, Abcam) primary antibody and subsequently incubated with matched secondary antibodies (#7074 and #7076, Cell signaling Technology, Beverly, MA, USA). Then, signals for each protein expression were detected with the Bio-Rad Gel imaging system (Bio-Rad, Hercules, CA, USA). GAPDH (G8140, US Biological, Swampscott, MA, USA) was used as a loading control. Alpha Innotech (San Leandro, CA, USA) imaging software was used to quantify western blotting data.