FPN is removed from the early phagosome and differs from TfR recycling. (A) The laser scanning confocal fluorescent micrographs depict RAW macrophages expressing FPN-GFP (top row) or TfR-GFP (bottom row) (both in green) and the PI(3)P biosensor 2×FYVE-RFP (red) that were allowed to phagocytose IgG-coated beads for 10 minutes. After fixation, the presence of phagosomal FPN-GFP, TfR-GFP, and 2×FYVE-RFP was analyzed. Arrows indicate a TfR and 2×FYVE-positive phagosome, whereas arrowheads indicate FPN-negative but 2×FYVE-positive phagosomes. Scale bars, 10 µm. (B) The graph depicts the fraction of 2×FYVE-positive phagosomes that are also positive for either FPN- or TfR-GFP. The data represent the mean ± SD derived from ≥56 phagosomes from at least 3 independent experiments. Statistical significance was determined using an unpaired Student t test with a Welch’s correction. (C) The confocal fluorescent micrographs also depict RAW macrophages expressing either FPN-GFP or TfR-GFP (in green) that were fixed and immunostained for endogenous EEA1 (in red) using rabbit anti-EEA1 antibody followed by anti-rabbit Cy3-conjugated secondary. The cells were allowed to phagocytose IgG opsonized beads for 10 minutes prior to fixation and staining. Scale bars, 10 μm. The white arrows point to phagosomes that are TfR positive and EEA1 positive, whereas the arrowheads point to representative phagosomes that are FPN negative yet are EEA1 positive. Scale bars, 10 μm. (D) The graph depicts the fraction of EEA1-positive phagosomes at 10 minutes post–bead exposure that are either TfR-GFP or FPN-GFP positive. The data are the mean ± SD of 3 independent experiments, and statistical significance was determined by an unpaired Student t test with a Welch’s correction.  **P < .01; ***P < .001.