Figure 5
Figure 5. 5-phosphatases regulate PtdIns(3,4)P2-association with forming phagosomes. RAW264.7 macrophages cotransfected with YFP-TAPP1/PH and (A) vector, (B) HA-72-5ptase, or (C) HA-72-D480N5ptase were stimulated with eIgG to initiate phagocytosis, and fluorescence was monitored using confocal microscopy. (A-C) YFP-TAPP1/PH expression is shown. Images represent a time course of phagocytosis of eIgG. The time of closure was arbitrarily designated as time 0 seconds. Arrowheads indicate sites of YFP-TAPP1/PH accumulation. Scale bar represents 20 μm. Insets show enlargements of boxed areas. The complete sequences from which these images are taken are shown in Videos S5, S6, and S7. (D) Results shown represent the fluorescence intensity of YFP-TAPP1/PH with phagosomes, normalized to the cytosolic fluorescence. To allow for comparison between experiments, each RFU was normalized to the maximum RFU obtained for each experiment. ---●--- represents vector; ▴, HA-72-5ptase; and ▒, HA-72-D480N5ptase. Data represent mean (± SEM) of 3 to 5 independent determinations. *P < .05 compared with vector-expressing cells. (E) The normalized fluorescence intensity of GFP-ARNO/PH and YFP-TAPP1/PH at the phagosomal membrane in cells coexpressing (i) vector, (ii) HA-72-5ptase, or (iii) HA-72-D480N5ptase were plotted on the same graphs for direct comparison. ○ represents YFP-TAPP1/PH, and ▒ represents GFP-ARNO/PH. *P < .05 compared with YFP-TAPP1/PH RFU. (F) siCON- or si72-5ptase-transfected RAW264.7 macrophages were further transfected with YFP-TAPP1/PH 48 hours after transfection and stimulated with eIgG or SOZ, and phagocytosis was monitored using confocal microscopy. Results shown represent the fluorescence intensity of GFP-ARNO/PH at phagosomes normalized to the cytosolic fluorescence. To allow for comparison between experiments, each RFU was normalized to the maximum RFU obtained for each experiment. • represents siCON, and ⧫ represents si72-5ptase. Data represent mean (± SEM) from 3 to 7 independent determinations. *P < .05 relative to siCON-expressing cells.

5-phosphatases regulate PtdIns(3,4)P2-association with forming phagosomes. RAW264.7 macrophages cotransfected with YFP-TAPP1/PH and (A) vector, (B) HA-72-5ptase, or (C) HA-72-D480N5ptase were stimulated with eIgG to initiate phagocytosis, and fluorescence was monitored using confocal microscopy. (A-C) YFP-TAPP1/PH expression is shown. Images represent a time course of phagocytosis of eIgG. The time of closure was arbitrarily designated as time 0 seconds. Arrowheads indicate sites of YFP-TAPP1/PH accumulation. Scale bar represents 20 μm. Insets show enlargements of boxed areas. The complete sequences from which these images are taken are shown in Videos S5, S6, and S7. (D) Results shown represent the fluorescence intensity of YFP-TAPP1/PH with phagosomes, normalized to the cytosolic fluorescence. To allow for comparison between experiments, each RFU was normalized to the maximum RFU obtained for each experiment. ---●--- represents vector; ▴, HA-72-5ptase; and ▒, HA-72-D480N5ptase. Data represent mean (± SEM) of 3 to 5 independent determinations. *P < .05 compared with vector-expressing cells. (E) The normalized fluorescence intensity of GFP-ARNO/PH and YFP-TAPP1/PH at the phagosomal membrane in cells coexpressing (i) vector, (ii) HA-72-5ptase, or (iii) HA-72-D480N5ptase were plotted on the same graphs for direct comparison. ○ represents YFP-TAPP1/PH, and ▒ represents GFP-ARNO/PH. *P < .05 compared with YFP-TAPP1/PH RFU. (F) siCON- or si72-5ptase-transfected RAW264.7 macrophages were further transfected with YFP-TAPP1/PH 48 hours after transfection and stimulated with eIgG or SOZ, and phagocytosis was monitored using confocal microscopy. Results shown represent the fluorescence intensity of GFP-ARNO/PH at phagosomes normalized to the cytosolic fluorescence. To allow for comparison between experiments, each RFU was normalized to the maximum RFU obtained for each experiment. • represents siCON, and ⧫ represents si72-5ptase. Data represent mean (± SEM) from 3 to 7 independent determinations. *P < .05 relative to siCON-expressing cells.

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