Odyssey clx

The total proteins of the liver were extracted, and the concentrations were measured by a BCA procedure. Samples containing 50 μg proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% bovine albumin (BSA) in Tris buffer saline-Tween 20 (TBST) for 2 h at 37°C, and then incubated overnight at 4°C with the respective first antibodies diluted in 1.5: 1000. After washing with TBST, the membranes were incubated with fluorescent secondary antibodies (1: 15000) for 2 h at 37°C. After rewashing with TBST, the membranes were scanned and visualized by a fluorescent scanner (Odyssey CLX, Gene Company Limited, United States).

SMMC-7721 cells (5 × 10⁴ cells/mL) were seeded on 90 × 20-mm dishes. After treatment, the SMMC-7721 cells were scraped off and washed twice with cold PBS. The cells were solubilized by RIPA lysis buffer (Beyotime, China) containing 1% phenyl methylsulphonylfluoride (PMSF, Beyotime, China) for 30 min on ice. Whole-cell lysates were clarified by centrifuging at 12 000 rpm for 15 min at 4 °C, and the supernatants were collected. Protein concentrations were determined by the BCA protein assay. Equal amounts of protein (50 μg) were separated by electrophoresis on 12% sodium dodecyl sulphate polyacrylamide gels and were transferred onto PVDF membranes. These membranes were soaked in 5% skimmed milk dissolved with TBST buffer (Tris Buffer Saline supplemented with 0.1% Tween-20) for 2 h to block nonspecific binding sites. The membranes were then incubated overnight at 4 °C with the primary antibodies (MMP2^{56 (link),57 (link)}, MYC^{58 (link),59 (link)}, Caspase3 and REG1A). After washing with TBST, the membranes were incubated for 2 h at room temperature with fluorescent secondary antibodies. After rewashing with TBST, the membranes were scanned using a fluorescent scanner (Odyssey CLX, Gene Company Limited, USA).