siRNA-mediated knockdown of AP-1 or AP-3 reduces plasma membrane localization and increases punctate distribution of HIV-2 Gag.

<p>(A-C) HeLa cells were harvested 42 hours post-transfection with 10 nM siRNA against AP-1γ, AP-2μ, AP-3δ, AP-1γ and AP-3δ, or negative control siRNA (-ve), or mock transfected (none). Western blots of cell lysates were probed with antibodies against AP-1γ (A), AP-2μ (B) and AP-3δ (C). The blots were also probed with an antibody against γ tubulin, as a control. In (B), the antibody that recognizes AP-2μ also recognizes AP-1μ, which serves as an additional control. Panels separated by a dotted line represent different parts of the same blot. (D, E) HeLa cells were co-transfected with HIV-1 (D) or HIV-2 (E) proviral plasmids and no siRNA, siRNAs against AP-1γ, AP-2μ or AP-3δ, or a negative control siRNA (-ve), and observed by confocal immunofluorescence microscopy. Quantification of the effect of siRNAs on Gag distribution is shown graphically (D and E). For each siRNA treatment, at least 200 cells were assessed from three independent experiments by two different people. Cells were scored as having predominantly plasma membrane (PM), punctate intracellular (punctate), or diffuse cytoplasmic (diffuse) distribution of Gag. Error bars represent the standard deviation and significant changes from no siRNA treatment are shown with an asterisk (p < 0.05).