Donkey anti rabbit rhodamine red fab

Huh7-S10-3 liver cells transfected with RNA transcripts of the genotype 3 HEV-P6 infectious clone were fixed at 5 days posttransfection using 80% acetone and blocked using 10% goat serum in PBST. The cells were then stained using rabbit anti-HEV ORF2 antibody (1:500 dilution) (46 (link)). The donkey anti-rabbit-Rhodamine Red Fab (1:2,000 dilution; The Jackson Laboratory, Maine, USA) was used as the secondary antibody. The nuclei were counterstained using DAPI.

293T cells, Huh7-S10-3 liver cells, and IPEC-J2 intestinal epithelial cells were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS), 1× antibacterial-antimycotic, and 1× minimal essential amino acid (Gibco-Thermo Fisher, MA, USA). The HEV genotype 3 Kernow P6 virus stock was prepared by transfecting Huh7-S10-3 cells with in vitro-transcribed capped RNA transcripts from the HEV Kernow P6 infectious clone. The following antibodies and siRNA were used in this study: anti-IRF3 (1:1,000; SCBT, CA, USA), anti-phospho-IRF3 (1:1,000; Millipore, MA, USA), anti-glyceraldehyde-3-phosphate dehydrogenase-horseradish peroxidase (anti-GAPDH-HRP) (1:5,000; Invitrogen, MA, USA), bovine-anti-rabbit-HRP (1:7,000; SCBT, CA, USA), donkey-anti-rabbit-rhodamine red Fab (1:2,000; Jackson Laboratory, ME, USA), and siRIG-I (20 μM; SCBT, CA, USA). The IFN-β-firefly luciferase plasmid and TK-Renilla luciferase plasmid for the IFN promoter assay were purchased from Promega (WI, USA). The pLIX-402 lentiviral vector and second-generation lentiviral packing vectors (psPAX2 and pMD2.G) were procured from Addgene (MA, USA).