Characterization of particles secreted by Huh-7.5 cells.

<p>(A) Analysis of HCV glycoprotein expression by immunofluorescence with E2-specific antibody. Huh-7.5 were grown under standard conditions and stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004233#s4" target="_blank">materials and methods</a>. Left panel, control cells, right panel, E1E2 transduced Huh-7. E2 positive cells are stained in red. (B) Density of apoB and E1E2 positive fractions in the E1E2-Huh-7.5 supernatant. Three day culture supernatant was run on a iodixanol gradient and fractions were analysed by apoB ELISA, upper panel, and western blotting with E1 and E2-specific antibodies, lower panel. Envelope control proteins were deposited in the last right lane (+). E1 and E2 proteins were only present in apoB positive fractions. (C) Immuno-precipitation of E2 positive particles was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004233#pone-0004233-g005" target="_blank">figure 5</a> with Huh-7.5 supernatant collected three days after seeding. Basal culture medium of control and E1E2-Huh-7.5 were collected after two days of culture in serum free medium. E2 positive particles were captured by E2-specific H48 monoclonal antibody and immuno-precipitated particles were analysed by western blotting and stained with H52 anti-E2 (upper panel), A4 anti-E1 (middle panel) and anti-apoB (lower panel) antibodies. Molecular weight are indicated on the left, control proteins were deposited in the + lane.