Depletion of PI[3]P fails to alter the phagosomal surface charge. (A and B) RAW macrophages were cotransfected with a construct consisting of two tandem PI[3]P-binding FYVE domains of EEA1 fused to mRFP (2FYVE-mRFP; red) and the GFP-5+ probe (green). (A) The cells were exposed to opsonized sRBCs, and confocal images were acquired after 15–20 min. (B) The cells were treated with wortmannin for an additional 15 min, and additional images were acquired. (C and D) Quantification of the ratio of fluorescence intensity of GFP-5+ on the phagosome versus the bulk plasma membrane (C) and the percentage of 2FYVE-mRFP–positive phagosomes (D) before and after wortmannin treatment. PM, plasma membrane. Images are representative of 20 fields from two similar independent experiments; quantification was performed on those same images. Data are means ± SEM quantified from at least 15 phagosomes from two experiments. Bars, 2 µm.