Interaction of M-PMV PR, HIV-1 PR and BCA3. (a) Western blot analysis of total lysates of the HEK-293 cells transfected with c-myc-M-PMV PR17(D26N), c-myc-M-PMV PR12(D26N), c-myc-HIV-1 PR(D25N) and HA-BCA3. The blots were developed with anti c-myc (upper panel) and HA (lower panel) antibodies. (b) Co-immunoprecipitation of M-PMV or HIV PRs with BCA3: HEK-293 (lanes 1–4) and HeLa (lanes 5–8) cells transfected with c-myc-M-PMV PR17(D26N), c-myc-M-PMVPR12(D26N) or c-myc-HIV-1 PR(D25N) together with HA-BCA3 were lysed 48 h post-transfection, and proteins were immunoprecipitated using anti-c-myc antibody. Precipitates were blotted and developed with anti-c-myc (upper panel) or anti-HA (lower panel) antibodies. (c) Immunocolocalization of HIV-1 PR(D25N) with BCA3 in HEK-293 cells using confocal microscopy: HEK-293 cells transfected with c-myc-HIV-1 PR(D25N) together with HA-BCA3 were stained 48 h post-transfection using anti HA-TRITC or c-myc-FITC antibodies and visualized using confocal microscopy. The two black panels represent the samples of the cells not producing BCA3 (first row) and HIV-1 PR (second row) demonstrating thus that HA-TRITC and c-myc-FITC antibodies do not provide non-specific signal.