Western Blot Analysis of HCC Cell Lines

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.