Fig. 3. Surface H72 and H161 proteins are captured in newly formed phagosomes. (A-C) Cells expressing CRAC-GFP (green) were allowed to engulf yeast particles (red) for 10 minutes. They were then fixed and processed for immunofluorescence using H72 or H161 antibodies. (A) Representative pictures of H72 labeling (white) are shown for shallow (1) and deep (2) phagocytic cups as well as newly formed (3) and maturing early (4) phagosomes. (B,C) Relative intensity of the H72 (B) or H161 (C) staining in each type of phagocytic structure (1, 2, 3 and 4). Each dot indicates the H72 (B) or H161 (C) fluorescence intensity in the membrane of one phagosome relative to the intensity at the cell surface in the same cell. A line indicates the average. (D,E) Cells were incubated with H72 or H161 antibodies for 5 minutes at 4°C, washed and allowed to engulf yeast particles (red) for 3 minutes. Cells were then fixed, permeabilized and incubated with a fluorescent secondary antibody (white). (D) Schematic representation of the experiment. (E) Surface H72 and H161 (white) are incorporated in newly formed phagosomes. Bars, 4 µm.