2.3. Transient Transfection and Western Blot Analysis

The transfection experiments and Western blot analysis are well established [12,16,17,19,21,22,23,24]. The human hepatoma cell line Huh7 was cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco). Cells were seeded into 6-well plates and transfected 18–24 h later, when the cell density reached about 80%. Plasmid DNA (2 μg/well) was transfected using Lipofectamine™ 3000 Transfection Reagent (Invitrogen). Cells and culture supernatant were harvested 72 h later. Cells were lysed in 120 μL lysis buffer consisting of 10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA; 1% NP40 and cOmplete™ protease inhibitor cocktail (Roche). Proteins from 1/15th cell lysate or precipitated from 100 μL of culture supernatant by 10% polyethylene glycol (PEG) were separated by SDS-polyacrylamide gel electrophoresis. Following transfer, the Western blots were incubated at 4 °C overnight with mouse monoclonal anti-preS1 (7H11; 1:4000), rabbit polyclonal anti-preS2 (GenScript, Shanghai, China; 1:2000), anti-HBs antibody (Novus, Centennial, CO, USA; 1:3000) or anti-HBc antibody (2A7; 1:10,000) diluted in 5% milk-TBST. The blots were washed for 30 min in TBST and incubated for 1 h with a 1:8000 dilution of goat anti-rabbit or anti-mouse antibody conjugated with horse radish peroxidase (HRP). The blots were washed for 30 min and signals were detected by Western Lightning Plus-ECL, Enhanced Chemiluminescence Substrate (Perkin-Elmer, Shelton, CT, USA). As a loading control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected by a mouse monoclonal antibody (Proteintech, 1:5000 dilution) followed by anti-mouse-HRP (1:10,000). Multi Gauge V2.2 software was used to measure the grey values of signals on the blots.