FIG. 9.
HIV-1 envelope gp120 associates with lipid rafts. (A) Jurkat T cells, untreated or treated with 10 mM MβCD, were incubated for 30 min on ice with recombinant HIV-1 gp120 IIIB (10 μg/ml). The cells were washed to remove unbound gp120 and then were exposed for 10 min at 37°C to rabbit anti-gp120 antibody to induce cross-linking. The cells were lysed in Triton X-100 buffer, and the postnuclear extracts were fractionated in a sucrose density gradient. An aliquot of each fraction (40 μl) was resolved on SDS-PAGE, and gp120 was visualized by Western blotting as described in Materials and Methods. A signal present in fraction 10 of the control (cells not exposed to gp120) represents nonspecific binding of polyclonal antibody. (B) Membrane-bound HIV-1 gp120 colocalizes preferentially with lipid rafts. Distribution and colocalization of the nonraft marker CD71 (transferrin receptor) and the raft marker GM1 with gp120 were analyzed by confocal microscopy as described in Materials and Methods.