Figure 2

Mitochondrial localization of HIV-1 PR and, BCA3. (a) HeLa cells were co-transfected with c-myc-HIV-1 PR(D25N), or HA-BCA3 and mitochondrial marker pDsRed2-Mito. HeLa cells were stained 48 h post-transfection using anti-HA-TRITC or c-myc-FITC antibodies and visualized using confocal microscopy. Mitochondria were stained red; the overlaid images show colocalization of mitochondrial marker with BCA3 and HIV-1 PR(D25N), as well as HIV-1 PR(D25N) with BCA3. (b) Purified mitochondria from HeLa cells transfected with c-myc-HIV-1 PR(D25N), HA-BCA3, or a combination of c-myc-HIV-1 PR(D25N) with HA-BCA3. Fractions from Optiprep gradient (labeled 1–12) were analyzed using Western blot with Mitochondrial Marker antibody and anti-HA and anti-c-myc antibodies. (c) Immunogold TEM analysis of localization of VDAC (left panels, bar represents 500 nm), BCA3 (middle panels, bar represents 500 nm) and HIV-1 PR(D25N) (right panels, bar represents 1 μm) in HEK-293 cells. (d,e) Western blot (d) and co-immunoprecipitation followed by Western blot analysis (e) of HIV-1 PR(D25N) and BCA3 present in cytosolic and mitochondrial fractions. HEK-293 cells (4x10⁵ cells/ml) were co-transfected with HA-BCA3 and c-myc-HIV-1 PR(D25N). After 48 h, the cells were washed 1× with PBS and 1/20 of the cells was resuspended in PLB and used as a total cell lysate sample for Western blot analysis (d, lane 1). 1/4 of the cells were used for immunoprecipitation (e, lane 4) using anti-HA and anti-c-myc antibodies. The last 3/4 of the cells was used for the isolation of cytosolic and mitochondrial fractions. 100 μl aliquots of both cytosolic and mitochondrial fractions were analyzed using Western blot (d, lanes 2 and 3, respectively) and 900 μl aliquots of both cytosolic and mitochondrial fractions samples were used for immunoprecipitation using anti-HA and anti-c-myc antibodies and developed by Western blot (e, lanes 5 and 6).