Figure 6
Absence of anti-DSB effect of zinc-binding domain mutants of Vif. Sf9 cells were coinfected with two baculoviruses at equal MOI of each (5 PFU/cell), one expressing Pr55Gag, the other expressing VifS116V (A and B, (i)) or VifC133S (A and B, (ii)). Cells were treated with increasing concentrations of DSB in DMSO for 30 h at 18 h pi, as indicated on top of panels (i) and (ii), and on the x-axis of panel (C). Cells were harvested at 48 h pi, and whole cell lysates (WCL) and extracellular VLP analyzed by SDS-PAGE and immunoblotting, using anti-Vif primary antibody and secondary peroxidase-labelled antibody, followed by anti-Gag primary antibody and phosphatase-labelled secondary antibody. (A), WCL; (B), VLP. (m), prestained molecular mass markers; (kDa), kiloDaltons. (C), Quantification of VLP produced by DSB-treated Sf9 cells coexpressing Pr55Gag and Vif mutants was performed using SDS-PAGE and autoradiography of immunoblots reacted with anti-Gag and 35S-labelled secondary anti-rabbit IgG antibody, as described in the legends to Fig. 3(c) and 5(c). Results were expressed as percentage of control, untreated samples, which was attributed the 100% value. Mean of three separate experiments ± standard deviation.