Mouse anti e1

Human embryonic kidney 293T (HEK293T) cells were cultured in 96 well plates in Dulbecco modified Eagle's medium (DMEM, Life Technologies) supplemented with 1% penicillin-streptomycin (Life Technologies) and 10% fetal calf serum at 37°C in 5% CO₂. The cells were transfected with 200 ng of DNA using Lipofectamine LTX (Life Technologies) according to the manufacturer's protocol. Thirty-six hours post-transfection, the cells were fixed in 4% paraformaldehyde then permeabilized with 100% methanol. The cells were then incubated with the appropriate primary antibody at 37°C for 2–4 h at 37°C or overnight at 4°C followed by the addition of the appropriate fluorophore-conjugated secondary antibody for 1 h at 37°C in the dark. Primary antibodies used include mouse anti-E1 (MyBiosource, Cat. No. MBS310203), goat anti-E2 (Virostat, Cat. No. 2851), pooled sera collected from vaccinated mice or human MAb (kindly supplied by Heidi Drummer). Secondary antibodies were Alexa Fluor (AF) 488-conjugated anti-mouse, AF488-conjugated anti-human and AF555-conjugated anti-goat (all from Invitrogen). Nuclei were stained with Hoechst 33342 (Life Technologies). Cells were visualized by fluorescence microscopy (Zeiss LSM-700) and the data digitized using the Zen software (Zeiss).

To confirm E1/E2 expression, supernatant fluids were harvested from HEK293T cells transfected with p-sE1, p-sE2, p-sE1E2, p-sE1-IMX313P, p-sE2-IMX313P, or p-sE1E2-IMX313P and 50 μg of protein was analyzed for antigen expression in 10–12% (v/v) SDS-PAGE under reducing or non-reducing conditions as described previously (55 (link)–57 (link), 59 (link), 62 (link)). Mouse anti-E1 (MyBiosource, Cat. No. MBS310203) and human anti-E2 MAb HCV1 were used as primary antibodies to detect E1 and E2 expression followed by followed by anti-mouse-AF488 and anti-human AF555 (both from Invitrogen). The membrane was imaged using the LAS4000 digital imaging system (Fujifilm) and the resultant images were overlaid using ImageJ software (National Institutes of Health, USA) to obtain colocalization data.