Anti svep1

Western blotting was conducted as described previously (Chen et al., 2017 (link); He et al., 2020 (link)).The following antibodies were used: anti-SVEP1 (1:500) from R&D (MAB97741), anti-GAPDH (1:1000) from Santa Cruz Biotechnology.

The fresh frozen HCC tissues were extracted with a tissue protein extractor. Western Blot was used to compare the SVEP1 expression level in tumor and para-tumor tissue. The cells were washed 3 times with cold PBS and lysed on ice for 30 min using SDS lysis buffer supplemented with 1 mM NaF, 1 mM Na3VO4, and 1x protease/phosphatase inhibitor cocktail. (Roche, Switzerland). The collected proteins were denatured at 95°C for 10 min and centrifuged at 12,000 rpm at 4°C about 10 min. Equal amounts of protein were loaded onto the gel and separated by SDS-PAGE. Then, transfer the protein to the PVDF membrane (Immobilon-P; Millipore, Billerica, MA, USA) and blocked with 5% skimmed milk or 3% bovine serum albumin (BSA), followed by incubation with primary and secondary antibodies. The following antibodies were used: anti-SVEP1 (1:250) from R&D SYSTEM; anti-GAPDH (1:1000) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Akt (1:1000), anti-Phospho-Akt (Thr308) (1:2000) and Phospho-Akt (Ser473) (1:2000) from Cell Signaling Technology (Beverly, USA)