Hrp conjugated goat anti rabbit or anti mouse igg antibody

Total proteins from cells were extracted using RIPA buffer (Cell Signaling Technology) supplemented with protease inhibitors and phosphatase inhibitors. Proteins of equal amounts (30 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking antigen, the membranes were incubated with primary antibodies and the corresponding secondary antibodies. Proteins were visualized with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Waltham, MA, USA). The primary antibodies used in this assay included E-cadherin, vimentin, N-cadherin, β-catenin, Lamin B1, GAPDH (Cell Signaling Technology), and APC (Abcam, Cambridge, UK). HRP-conjugated goat anti-rabbit or anti-mouse IgG antibody (Abcam) was used as the secondary antibody. For extraction of cytoplasmic and nuclear proteins, BeyoECL Plus Nuclear and Cytoplasmic Protein Extraction Kits (Beyotime Institute of Biotechnology, Jiangsu, China) were utilized according to the manufacturer’s instructions.

The procedure of Western blot was performed as previously described [33 (link)]. Briefly, HCC cells were collected and lysed in RIPA lysis buffer with protease inhibitor (Selleck, Houston, USA) on the ice. After quantification, 30 μg of lysate protein were separated by SDS-PAGE gel and then transferred onto PVDF membrane. After blocking nonspecific binding sites with 5% non-fat milk, the membranes were incubated with primary antibodies at 4°C overnight. The proteins in the membranes were visualized with the SuperSignal® ECL Kit (Pierce, USA). The primary antibodies used in this study included GAPDH, E-cadherin, Vimentin, Fibronectin (Cell Signaling Technology, USA), NFASC (Proteintech, Wuhan, China) and MMP-9 (Santa Cruz, USA). HRP-conjugated goat anti-rabbit or anti-mouse IgG antibody (Abcam, Cambridge, UK) was used as the secondary antibody. GAPDH was used as a loading control.

Total lysates were obtained from harvested ESCC cells with the 1 × RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with protease inhibitors. The concentration was determined by the BCA Protein Assay kit (Pierce, Rockford, IL, USA) and identical quantities of proteins (20–30 μg) were separated by SDS-polyacrylamide gel electrophoresis. The proteins were then transferred to PVDF membranes. After blocking with 5% non-fat milk in TBST, the membranes were probed with the indicated primary antibodies at 4 °C overnight with gentle shaking. The membranes were then exposed to the corresponding secondary antibody and the SupreSignal ECL Kit (Pierce) was used to detect bands, with GAPDH as the internal control. The primary antibodies used in our study include E-cadherin (1:500), Vimentin (1:500), β-catenin (1:500, Cell Signaling Technology), Kindlin-2 (1:2000, Millipore, Bedford, MA, USA) p-FAK(Tyr397) (1:1000, Cell Signaling Technology), FAK (1:500, Cell Signaling Technology), active RhoA (1:500, Cell Signaling Technology), total RhoA (1:500, Cell Signaling Technology) and GAPDH (1:5000, Sigma, Sigma-Aldrich, St. Louis, MO, USA). HRP-conjugated goat anti-rabbit or anti-mouse IgG antibody (Abcam, Cambridge, MA, USA) was used as the secondary antibody.