FIG. 3.

Effect of wortmannin on fusion of nascent phagosomes with early endosomes. (A to F) CHO-IIA cells that were untreated (A to C) or pretreated with wortmannin (D to F) were allowed to internalize IgG-opsonized latex beads for 20 min. After washing excess beads, the cells were incubated briefly on ice with anti-human antibody conjugated with fluorescein isothiocyanate to identify adherent, incompletely internalized beads (B and E). The cells were then incubated for an additional 10 min at 37°C with FM4-64.The red fluorescence of FM4-64 is shown in A and D (arrows). C and F are the differential interference contrast images corresponding to A and D, respectively. (G to I) CHO-IIA cells transiently transfected with syntaxin 13-GFP were treated with wortmannin (H and black columns in I) or left untreated (G and empty columns in I) and allowed to internalize particles for 20 min. Adherent, incompletely internalized beads were then stained as above using Texas red-conjugated antibodies. Lastly, the cells were chased for the indicated times and fixed, and confocal fluorescence microscopy was used to visualize the distribution of syntaxin 13. (G and H) representative fluorescence images, with corresponding differential interference contrast insets. Arrows point to phagosomes. Scale bars =10 μm. (I) Quantitation of the effect of wortmannin on syntaxin 13 acquisition by phagosomes. Data are means + SE (error bars) of three separate experiments, each with at least 100 individual phagosomes from 30 to 50 different cells counted.