Figure 3
Figure 3. Intracellular localization of 72-5ptase during phagocytosis. (A) RAW264.7 macrophages grown in serum or serum-starved and stimulated with 3-bIgG for 7 minutes were fixed and labeled with 72-5ptase (red) or immunodepleted-72-5ptase-specific antibodies and colocalized with phalloidin (green). Arrowheads indicate sites of 72-5ptase localization, “b” indicates 3-bIgG, and asterisks represent fully internalized beads. Scale bar represents 10 μm. (B) RAW264.7 cells transfected with HA-72-5ptase or HA-SHIP1 were stimulated with eIgG for 7 minutes, fixed, and labeled with HA antibodies (red) and phalloidin (green). Scale bar represents 10 μm; “e” indicates eIgG. (C) RAW264.7 transfected with GFP-72-D480N5ptase were stimulated with eIgG to initiate phagocytosis, and fluorescence was monitored using confocal microscopy. Images represent a time course of phagocytosis of eIgG. The time of closure was arbitrarily chosen as time 0 seconds. Arrowheads indicate sites of GFP-72-D480N5ptase accumulation, and asterisks indicate phagosomes forming parallel to the coverslip. Scale bar represents 20 μm. The complete sequence from which these images were taken is shown in Video S1. (D) To characterize the temporal and spatial association of GFP-72-D480N5ptase with phagosomes, the accumulation of fluorescence at the phagocytic cup normalized to the cytosolic level (RFU) was quantified. To allow for comparison between experiments the RFUs were normalized to the maximum RFU obtained for each experiment. Data are expressed as the mean (± SEM) of 3 independent experiments. (E) RAW264.7 cells transfected with HA-72-5ptase or HA-SHIP1 were stimulated with SOZ for 10 minutes, fixed, and labeled with HA antibodies (red) and phalloidin (green). “SOZ” indicates serum-opsonized zymosan. Scale bar represents 10 μm. (F) RAW264.7 macrophages stimulated with 6-bIgG or SOZ for 7 and 10 minutes, respectively, were fixed and labeled with 72-5ptase (red)– and SHIP1 (green)–specific antibodies. Arrowheads indicate protein localization, “b” indicates 6-bIgG, and “SOZ” indicates SOZ. Scale bar represents 10 μm. Insets show enlargements of boxed areas.

Intracellular localization of 72-5ptase during phagocytosis. (A) RAW264.7 macrophages grown in serum or serum-starved and stimulated with 3-bIgG for 7 minutes were fixed and labeled with 72-5ptase (red) or immunodepleted-72-5ptase-specific antibodies and colocalized with phalloidin (green). Arrowheads indicate sites of 72-5ptase localization, “b” indicates 3-bIgG, and asterisks represent fully internalized beads. Scale bar represents 10 μm. (B) RAW264.7 cells transfected with HA-72-5ptase or HA-SHIP1 were stimulated with eIgG for 7 minutes, fixed, and labeled with HA antibodies (red) and phalloidin (green). Scale bar represents 10 μm; “e” indicates eIgG. (C) RAW264.7 transfected with GFP-72-D480N5ptase were stimulated with eIgG to initiate phagocytosis, and fluorescence was monitored using confocal microscopy. Images represent a time course of phagocytosis of eIgG. The time of closure was arbitrarily chosen as time 0 seconds. Arrowheads indicate sites of GFP-72-D480N5ptase accumulation, and asterisks indicate phagosomes forming parallel to the coverslip. Scale bar represents 20 μm. The complete sequence from which these images were taken is shown in Video S1. (D) To characterize the temporal and spatial association of GFP-72-D480N5ptase with phagosomes, the accumulation of fluorescence at the phagocytic cup normalized to the cytosolic level (RFU) was quantified. To allow for comparison between experiments the RFUs were normalized to the maximum RFU obtained for each experiment. Data are expressed as the mean (± SEM) of 3 independent experiments. (E) RAW264.7 cells transfected with HA-72-5ptase or HA-SHIP1 were stimulated with SOZ for 10 minutes, fixed, and labeled with HA antibodies (red) and phalloidin (green). “SOZ” indicates serum-opsonized zymosan. Scale bar represents 10 μm. (F) RAW264.7 macrophages stimulated with 6-bIgG or SOZ for 7 and 10 minutes, respectively, were fixed and labeled with 72-5ptase (red)– and SHIP1 (green)–specific antibodies. Arrowheads indicate protein localization, “b” indicates 6-bIgG, and “SOZ” indicates SOZ. Scale bar represents 10 μm. Insets show enlargements of boxed areas.

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