Zd409

Protein extraction from HCC cells was conducted using protein lysis buffer (ZD409, ZOMANBIO, Beijing, China) and a Total Protein Extraction Kit (BC3711, Solarbio, Beijing, China). The obtained proteins were separated via SDS–PAGE (P1200, Solarbio) and then transferred onto PVDF membranes (T2234, Thermo Fisher). Following blocking with 5% skimmed milk, the membranes were incubated with primary antibodies. Following this, the membranes underwent incubation with secondary antibodies for 1 h. Protein levels were determined using an enhanced chemiluminescence detection system, with β‐actin serving as the internal reference.

Protein lysis buffer (ZD409, ZOMANBIO, Beijing, China) and a Total Protein Extraction Kit (BC3711, Solarbio, Beijing, China) were employed to extract total proteins from HCC cells. The obtained proteins were isolated by SDS–PAGE (P1200, Solarbio) and then transferred onto PVDF membranes (T2234, Thermo Fisher). After blocking with 5% skimmed milk, the membranes were cocultured with primary antibodies, including anti-HIF-1α (ab1, Abcam), anti-β-actin (ab179467, Abcam), anti-MMP2 (ab92536, Abcam), anti-MMP9 (ab76003, Abcam), anti-ZO-1 (ab276131, Abcam), anti-E-cadherin (ab40772, Abcam), anti-N-cadherin (ab76011, Abcam), anti-vimentin (ab92547, Abcam), anti-Ac-K (ab190479, Abcam), anti-FOXA3 (PA1-813, Abcam), anti-HDAC2 (ab32117, Abcam), anti-PKM2 (ab137791, Abcam) and anti-DADACT3 (ab797, Abcam). Subsequently, the membranes were incubated with secondary antibody for 1 h. The protein levels were measured by means of an enhanced chemiluminescence detection system. β-actin served as the internal reference.