Western Blot Analysis of EMT Markers

The cell extracts were lysed with ice-cold lysis buffer. The protein concentration was calculated using BCA Protein Assay kit (23,227, Pierce) and equal amount of proteins were separated by SDS-PAGE, as described previously [17 (link)]. Then, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane which was blocked for 1 h with 5% nonfat milk and incubated with primary antibodies against Matrix metalloproteinase (MMP)2 (1:1000, 74kD, ab215986, Abcam), MMP9 (1:1000, 78kD, ab219372, Abcam), E-cadherin (1:10,000, 97kD, ab40772, Abcam), N-cadherin (1:1000, 130kD, ab18203, Abcam) and GAPDH (1:10,000, 36kD, ab181602, Abcam) at 4°C overnight, followed by the incubation with secondary antibody goat anti-rabbit IgG H&L (1:1000, ab205718, Abcam) for 1 h. Then, the Image-Pro Plus 6.0 software (Media Cybernetics, Inc., MD, USA) was used to analyze proteins expressions. GAPDH was used as an internal control.