Anti zeb1

The proteins isolated from cells were separated using 10% SDS-PAGE and then transmitted to nitrocellulose (NC) membranes (Sigma, San Francisco, CA, USA). After that, the membranes were incubated with primary antibodies overnight, followed by incubation of horseradish peroxidase (HRP)-conjugated secondary antibodies (no. ab6728, 1:5,000; Abcam, UK) for another 1 h. The primary antibodies used in this study are as follows: anti-E-cadherin (no. 14472, 1:1,000), anti-β-catenin (no. 8480, 1:1,000), anti-N-cadherin (no. 13116, 1:1,000), anti-hnRNP-K (no. 9081, 1:1,000), anti-YAP (no. 14074, 1:1,000), anti-phospho-YAP (Ser¹²⁷) (no. 13008, 1:1,000), anti-TAZ (no. 70148, 1:1,000), anti-phospho-TAZ (Ser⁸⁹) (no. 59971, 1:1,000) (all of the above were purchased from Cell Signaling Technology, Boston, MA, USA); anti-Vimentin (sc-66002, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-ZEB1 (A301-921A, 1:2,000; Bethyl Laboratories, USA); and anti-GAPDH (G5262, 1:1,000; Sigma-Aldrich). GAPDH served as a normalized control, and proteins were captured using SuperSignal chemiluminescence substrate (Pierce, Thermo Scientific, Waltham, MA, USA) and analyzed by ImageJ (National Institutes of Health, USA).

SDS lysis buffer (0.05 mM Tris-HCl, 50 mM BME, 2% SDS, 0.1% Bromophenol blue, 10% glycerol) was used to collect protein from cells. Samples were heat denatured. Protein was equally loaded, separated on a 10% SDS-page gel, transferred onto a pure nitrocellulose membrane (BioRad), and blocked with 5% milk. Primary antibodies for immunoblotting included anti-pSMAD2 (1:1000, Cell Signaling, 3108), anti-pSMAD3 (1:250, ThermoFisher, 44-246 G), anti-SMAD2/3 (1:1000, Cell Signaling, 3102), anti-CDH1 (1:5000, BD Biosciences, 610181), anti-CDH2 (1:2000, BD Biosciences, 610920), anti-ZEB1 (1:3000, Bethyl, A301-922A), anti-Vimentin (1:5000, BD Biosciences, 550513), and anti-β-Actin (1:5000, Abcam, ab8227) for loading control. Membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1:5000, GE Healthcare) or anti-rabbit secondary antibody (1:5000, GE Healthcare) for 1 h, after which chemiluminescent signals were detected using ECL substrate (GE Healthcare).