FIG. 3.

Fractionation of cytoplasmic extracts of HIV-1-infected H9 cells by using sucrose density gradients. (A) Densities of fractions from a typical sucrose gradient, in this case the gradient analyzed in panel B. H9 cells transiently infected with HIV-1 (B) or HIV-1/Δvif (C) and uninfected H9/hVif cells (D) were lysed with PBS–1% TX-100. In addition, H9/hVif cells were also lysed in the absence of detergent by nitrogen cavitation (E). The postnuclear supernatant from each lysate was loaded onto a 20 to 60% (wt/vol) continuous sucrose gradient and centrifuged at 150,000 × g for 2 h. Ten fractions were harvested (1 = top; 10 = bottom), diluted and centrifuged to pellet the high-molecular-mass complexes. Equivalent amounts from each fraction were resolved on SDS-polyacrylamide gels and analyzed by Western blotting using antibodies specific for CA or Vif.