PS is required for the targeting of Src to the phagosome. (A and B) RAW macrophages cotransfected with c-Src–GFP (green) and the PS biosensor mRFP-Lact-C2 (red; A) or cotransfected with c-Src–GFP (green) and the PI[3]P biosensor mRFP-PX (red; B). The cells were exposed to IgG-opsonized sRBCs, and confocal images were acquired 1 h after initiation of phagocytosis. Asterisks denote phagosomes. (C–E) RAW macrophages transfected with c-Src and cotransfected with mRFP-Lact-C2 (D) or GFP-5+ (E) were infected with C. trachomatis elementary bodies for 4 (C) or 18 h (D and E). Cells in C were stained with anti–C. trachomatis antibodies, whereas those in D and E were incubated with the DNA stain Draq5 to identify the C. trachomatis invasion vacuoles (x). N indicates the location of the macrophage nucleus. Insets show a magnified C. trachomatis elementary body at 4 h as marked by the box. Images in A–E are representative of at least 30 cells from two similar experiments. (F) Quantification of the percentage of C. trachomatis inclusion vacuoles that bound c-Src at 6 and 18 h. (G) Quantification of the percentage of C. trachomatis inclusion vacuoles that bound the 5+ probe. Data in F and G are means ± SEM quantified from at least 35 phagosomes from two similar experiments. Bars, 2 µm.