PDI fails to segregate in GM1 and TX-100–resistant rafts. (A) Sup T1 cells were treated in the presence or absence of gp120 then incubated with anti-PDI rabbit PAb and cholera toxin B subunit coupled to Alexa Fluor 594 for 45 minutes at 4°C. Raft aggregation was induced by incubation of cholera toxin-labeled cells in the presence of antirabbit Cy2-coupled Ab for 10 minutes at 37°C. Cells were then fixed, attached to poly-l-lysine slide, mounted, and analyzed by confocal microscopy. The data shown are representative of the staining detected in more than 90% analyzed cells. Original magnification, × 63. (B) Shown is glycerol gradient distribution of nonraft (TfR) and raft (SrcK) markers in cells lysed with 0.25% TX-100 (panels i-ii), cells lysed by hypotonic lysis without TX-100 (panels iii-iv), cells in which cholesterol was depleted with 10 mM MβCD followed by lysis in the presence of TX-100 (panels v-vi). Glycerol gradient distribution of PDI and gp120 in samples lysed with 0.25% TX-100 is shown in panels vii to ix. Cells in panel h were pretreated with soluble gp120 III_B prior to detergent lysis. Data shown are representative of 4 experiments.