Western blot analysis

The HepG2 cells were seeded until the cell density reached 80%, and then cultured with 10 μM of the tested compounds for 24 h at 37 °C. After for 24 h, cells were collected, centrifuged, and washed twice with ice-cold PBS. And then, the cells were lysed in cell lysis buffer containing PMSF for 30 min, and lysates were collected by centrifugation at 4 °C for 10 min. The concentration of protein was measured by the BCA (bicinchoninic acid) protein assay reagents. Equal amounts of protein per line were separated on 12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF Hybond-P membrane (GE Healthcare). Membranes were incubated with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) buffer for 1 h, and then, the membranes being gently rotated overnight at 4 °C. Membranes were then incubated with primary antibodies at 4 °C overnight. Next, membranes incubated with peroxidase-labeled secondary antibodies for 2 h at 25 °C. The protein blots were detected by chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). The β-actin was used as the loading control.