Anti zeb1 antibody

The Western blotting analysis was performed using standard procedures. The following primary antibodies were used in the experiments: anti-PTBP1 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-PTBP2 antibody (Abcam, Cambridge, UK), anti-PTBP3 antibody (Sigma-Aldrich, St. Louis, MO, USA), anti-HNRNPK antibody (Abcam), anti-HNRNPM antibody (Sigma-Aldrich), anti-FUBP3 antibody (Abcam), anti-CPSF2 antibody (Abcam), anti-G3BP2 antibody (Atlas Antibodies), anti-TGFβ1 antibody (Proteintech Group), anti-TGFβ2 antibody (Abcam), anti-p-SMAD2 antibody (Cell Signaling Technology), anti-SMAD2 antibody (Cell Signaling Technology), anti-p-SMAD3 antibody (Cell Signaling Technology), anti-SMAD3 antibody (Cell Signaling Technology), anti-SMAD5 antibody (Abcam), anti-ID2 antibody (Abcam), anti-ZEB1 antibody (Abcam), anti-SNAI antibody (Abcam), anti-E-cadherin antibody (Proteintech Group), anti-Vimentin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Cell Signaling Technology) and anti-GAPDH antibody (Sigma-Aldrich). The blots were incubated with a goat anti-rabbit or anti-mouse secondary antibody (Sigma-Aldrich) and visualized with a commercial ECL kit (Pierce, Rockford, IL).

Cell total RNA was extracted with Trizol reagent (Invitrogen) and cDNA was synthesized from at least 3μg of total RNA using oligo (dT) and random hexamer primers. All primers (synthesized by GenePharma) used for RT–qPCR were listed in the Supplementary Table 3, and qPCR settings were 94°C for 2 min followed by 35 cycles of 94°C 15 s, 56°C 20 s and 72°C 30 s and then followed by 72°C for 2 min.
Cell total proteins were obtained by homogenization in 2× loading buffer, resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to western blot with corresponding antibodies. Anti-MZF1 and anti-SOX10 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-ZEB1 antibody was purchased from Abcam (Cambridge, MA).

The ChIP assays were performed using a Chromatin Immunoprecipitation Assay Kit (Millipore, Bedford, MA, USA). MKN28 cells were exposed to TGFβ1 or vehicle for 24 h and then crosslinked, lysed and sonicated. Immunoprecipitation was performed using an anti-ZEB1 antibody (Abcam, Cambridge, UK) and IgG. The precipitated DNA was quantified using qPCR and normalized to the respective 2% input.