Mab8002

JEG-3 cells were maintained in MEM supplemented with 10% FBS in the cell incubator with 5% CO₂ at 37°C. Cells were inoculated with 1 × 10⁵ copies HEV when they had grown to 60–70% in 35 mm glassy bottom dishes, continued to co-culture with HEV for 24 h. The medium was discarded and the cells were washed with cold PBS for 3 times. 4% paraformaldehyde was added to cells and fixed for 15 min. After washing, serum was added to the cells and incubated for 30 min to block antigens. The cells were incubated with a mouse anti-HEV monoclonal antibody (Millipore, MAB8002, 1:200) at 4°C overnight. The next day, after washing with PBS, the cells were incubated with goat anti-mouse IgG H&L (Alexa Fluor^® 647) for 45 min at room temperature. After the supernatant discarded and the dishes naturally air-dried, antifade mounting medium which was provided by Beyotime Biotechnology (Shanghai, China) was added to the cells. Fluorescence microscope, the instrument provided by Leica (Heidelberg, Germany) was operated to observe and take photos. The cell culture medium, serum, antibodies, and other materials used in this part have been mentioned before.

Cells were lysed in lysis buffer (25 mM Tris HCl pH 8.8, 50 mM NaCl, 0.5% Nonidet P-40 and 0.1% sodium dodecyl sulphate supplemented with cocktails of protease and phosphatase inhibitors). Debris were removed by centrifugation at 16,000 g for 20 min at 4°C. Total protein concentration was then determined using a Micro BCA^TM Protein assay (Thermo Fisher Scientific, Waltham, MA, USA). Equal amount of protein lysate was heat-treated in loading sample buffer containing β-mercaptoethanol and analysed by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to nitrocellulose membrane (Hybond-ECL, Amersham, GE healthcare LifeScience, Pittsburgh, PA, USA). Membranes were covered with blocking buffer (PBS containing 5% dry milk and 0.05% Tween-20) for one hour and then incubated with a mouse anti-HEV ORF2 antibody (MAB8002, Merck Millipore, Darmstadt, Germany) or a mouse anti-actin antibody (clone AC-40, Sigma-Aldrich, Saint-Louis, MO, USA) diluted in blocking buffer (1/500 and 1/2000 dilution, respectively). After several washes in PBS containing 0.05% Tween-20, a horseradish peroxidase-conjugated anti-mouse secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) was added (dilution 1/5000 in blocking buffer). An enhanced luminol-based chemiluminescent detection system was used to detect bound antibodies.