Western Blot Analysis of EMT Markers

The cell lysates preparation using the methods as previously described (15 (link), 16 (link)). The cell extracts were separated by 8 or 10% polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in TBS-T buffer (20 mM Tris, 137 mM NaCl, pH 7.4) with 5% nonfat milk for 1 h, and the blots were probed with the indicated primary antibodies for 24 h at 4°C. After washing steps with TBS-T (10 min each) for three times, the blots were incubated with horseradish peroxidase anti-mouse IgG or goat anti-rabbit for 1 h at room temperature. Last, the blots were visualized by ECL reagent (Millipore), and the protein expression was detected by chemiluminescence. The relative photographic density was quantified by ImageQuant LAS 4000 mini Biomolecular Imager (GE Healthcare Bio-Sciences AB, Björkgatan 30 751 84 Uppsala, Sweden). EMT antibodies for E-cadherin [(24E10) Rabbit mAb #3195], Vimentin [(D21H3) XP® Rabbit mAb #5741], TCF8/ZEB1 [(D80D3) Rabbit mAb #3396], Snail-1 [(Rabbit mAb #3879), Slug [(C19G7) Rabbit mAb #9585], Caludin-1 [(D5H1D) XP® Rabbit mAb #13255], β-catenin [(D10A8) XP^® Rabbit mAb #8480] were obtained from Cell Signaling; antibodies for EMT were obtained from Cell Signaling EMT antibody sampler kit #9782. Twist (GTX127310) antibody was obtained from GeneTex (Hsinchu City, Taiwan).