Functionally targeted SIV Gag protein to lysosomes and MHC II compartments using confocal microscopy.

<p><b>A</b>, HeLa cells were transfected with pVAX-SIVgag plasmid (top) or pVAX-SIVgag-LC3 plasmid (bottom) with chloroquine (CQ) treatment, and then stained with mouse anti-SIVgag IgG antibody and rabbit anti-LAMP-2 IgG antibody. Subsequently, Cy3-labeled goat anti-mouse IgG (red fluorescence) and Alexa Fluor 488-labeled goat anti-rabbit IgG (green fluorescence) secondary antibodies were used; the nuclei were stained with DAPI (blue fluorescence). The scale bar represents 20 μm. The data represent three independent experiments. <b>B</b>, RAW 264.7 cells were stimulated with lipopolysaccharide(LPS) and infected with Ad5-SIVgag (top) or Ad5-SIVgag-LC3 plasmid (bottom) with chloroquine (CQ) treatment, and subsequently stained with mouse anti-SIVgag IgG antibody and rat anti-MHC- II IgG antibody. Next, Cy3-labeled goat anti-rat IgG (red fluorescence) and 488-labeled goat anti-mouse IgG (green fluorescence) secondary antibodies were used; the nuclei were stained with DAPI (blue fluorescence). The scale bar represents 20 μm. The data represent three independent experiments. <b>C</b>, HeLa cells were transfected with pVAX, pVAX-SIVgag or pVAX-SIVgag-LC3b for 24 h, and then treated with 75 μM CQ or 200 nM RAPA for 24 h. LC3b-I, LC3b-II and SIVgag were visualized using anti-LC3b and anti-SIVgag immunoblotting, respectively. GAPDH blots demonstrate that CQ or RAPA treatment did not affect the overall protein level. D, SIVgag antigen-specific CD4+ T cells were obtained and isolated using anti-CD4 microbeads, and purified CD4+ T cells (effector cells) were then co-cultured with BMDC cells (target cells). The E:T ratios were 3∶1 or 9∶1. After 48 h, the culture supernatants were measured using the IFN-γ ELISA kit. As a positive control, the BMDC cells stimulated with LPS (1 μg/ml) were used as a positive control, and untreated DMDC cells were used as a negative control. These data were expressed as the mean±SEM from four mice samples.