Anti zeb2

Immunoblotting was performed as described previously [7] (link). Anti-JARID2 (#NB100-2214, Novus Biologicals), anti-E-cadherin (#610181, BD Transduction Lab), anti-Fibronectin (SAB4500974, Sigma), anti-Vimentin (ab8069, Abcam), anti-ZEB1 (#3396, Cell Signaling), anti-ZEB2 (#61096, Active Motif), anti-phosphorylated SMAD3 (ab51451, Abcam) and anti-GAPDH (6C5, Millipore) antibodies were used. To detect the morphological changes of the cells, A549 or HT29 cells were stained with 0.4% crystal violet (Waldeck). To allow direct fluorescence of actin cytoskeleton, the cells were stained with 0.25 µM tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (Sigma). For indirect immunofluorescence, the specimens were incubated with anti-E-cadherin antibody and treated with Alexa546-conjugated anti-mouse IgG antibody (Invitrogen). Nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI).

Whole-cell lysates were prepared using RIPA buffer as described previously [19 (link)] and analyzed using the following primary antibodies: anti-uPA, anti-c-Src, anti-ZEB1, anti-β-actin, and anti-GAPDH (Santa Cruz Biotechnology); anti-vimentin (Sigma, St Louis, MO, USA); anti-myc (Upstate Biotechnology, Lake Placid, NY, USA); anti-Slug, anti-Snail, anti-cyclin D1, anti-phospho-c-Jun(S63), anti-c-Jun, anti-phospho-ATF-2(T71), anti-phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), anti-ERK1/2, anti-phospho-c-Src(Y416), anti-phospho-JNK(T183/Y185), and anti-JNK (Cell Signaling Tech., Danvers, MA, USA); anti-Twist1 (Abcam, Cambridge, MA, USA); anti-ZEB2 (Active Motif, Tokyo, Japan); anti-Axl (R&D systems, Minneapolis, MN, USA); anti-TMPRSS4 (in-house) [19 (link)]. Where indicated, cells were transiently transfected for 24 h and then treated with 40 mM PD98059, 15 mM SP600125, 3 mM SU6656 (Sigma), or 0.4% dimethyl sulfoxide (DMSO) for 24 h before lysate preparation.