Figure 2

IN-BAD is efficiently biotinylated in producer cells and incorporated into virions. IN-BAD (lanes 1, 3) or IN-WT (lanes 2, 4) vector particles (30 ng of p24^gag) were either untreated (lanes 1, 2) or incubated with streptavidin paramagnetic beads and eluted (SA capture, lanes 3, 4). Samples were run on SDS-PAGE and Western blots (WB) were analysed with anti-IN (top) or anti-biotin (bottom) antibodies (1 minute exposure). Supernatants (spnt) from SA captures were also analysed (lane 5 and 6). (B) Left panel: streptavidin paramagnetic beads capture (SA capture) of the biotinylated IN (IN-BAD) from extracts of 293 cells mock-transduced (Mock) or transduced with the IN-BAD vector (293 IN-BADv), analysed by Western blotting with an anti-IN antibody. Middle panels: as controls, MA or p24 were immunoprecipitated (IP) respectively with an anti-MA and an anti p24 antibodies from the same cells extracts and analysed by WB respectively with the same antibodies. Right panel: HA tagged integrase (IN-HA) was immunoprecipitated with an anti-HA antibody from lysed IN-HA vector (IN-HAv) or from extracts of 293 cells mock-transduced (Mock) or transduced with IN-HAv (293 IN-HAv) and analysed by Western blotting with an anti-IN antibody. (C) Streptavidin paramagnetic beads capture of the biotinylated MA (MA-BAD) from extracts of 293 cells mock-transduced (Mock) or transduced with the MA-BAD vector (293 MA-BADv), or from lysed MA-BAD vector (MA-BADv) analysed by Western blotting with an anti-IN antibody.