Figure 2.
Effects of wortmannin on phagosomal acquisition of PI(3)P, EEA1, and LAMP-1. RAW cells were either left untreated (A, C, and open symbols in E) or pretreated with 100 nM wortmannin for 30 min (B, D, and solid symbols in E), before exposure to IgG-opsonized latex beads (3 μm diam). In A and B, the cells were transfected with 2FYVE–GFP and allowed to internalize beads for 7 min before fixation. In C and D, phagocytosis was allowed to occur for 7 min, followed by an additional 30 min after washing unbound beads. Subsequently, cells were immunostained for LAMP-1. Confocal fluorescence microscopy is shown in the main panels and DIC in the insets. Bars, 10 μm. (E) Quantification of the effect of wortmannin on the fraction of phagosomes containing EEA1 (triangles) or LAMP-1 (squares). Cells were pretreated with or without wortmannin and allowed to internalize beads for 7 min, as above. After washing unbound beads, incubation at 37°C proceeded for the indicated times, followed by immunostaining with antibodies to EEA1 or LAMP-1. Data are means ± SE of five experiments, each with at least 100 phagosomes counted.