Amersham ecl plus

5 mL of HepG-2 cells (1 × 10⁵ cells per mL) were seeded on a 100 mm diameter dish and incubated for 24 h at 37 °C, in 5% CO₂/95% air atmosphere. Cells were harvested and washed 3 times with ice-cold PBS after treatment of L6 (5 μM) or C6 (5 μM) for 24 hours. The cells were disrupted with buffer and centrifuged at 10 000g for 15 minutes under 4 °C to obtain a supernatant. The protein concentration was determined using a BSA Assay Kit (Beyotime, China), and the samples were detected by SDS-polyacrylamide gel electrophoresis. Under ice bath conditions, the protein was loaded on the polyvinylidene fluoride membrane for 3 hours, followed by preparation of skim milk (5%) for the presence of protein-blocked 1 h in TBST buffer at 25 °C. Membranes and primary antibodies were incubated overnight at 4 °C and washed three times with TBST buffer (20 mM Tris–HCl, 0.05% Tween 20, 150 mM NaCl, pH 8.0). After the membranes were incubated with the secondary antibody for 1 h at 25 °C, they were washed three times in the same manner. Immunoreactivity was imaged by Amersham ECL Plus (Amersham) and the amount of protein in each lane was determined by β-actin.