PtdIns(3,4)P 2 and its effector protein Lpd co-localize at the site of particle engagement during phagocytosis. (A) Representative confocal sections of RAW 264.7 macrophages co-expressing mCherry-cPHx3 and GFP-Lpd during phagocytosis. Asterisks denote sites of phagocytic cup formation. (B) Representative confocal extended-focus projection of RAW 264.7 macrophages co-expressing mCherry-cPHx3 and GFP-Lpd during phagocytosis following pre-treatment with 100 nM wortmannin. Asterisks represent sites of particle engagement. Inset shows engaged SRBCs. (C) Normalized phagosomal Lpd fluorescence intensity in control (n = 62) and wortmannin-treated cells (n = 82). Unpaired t test of individual values across n = 3 independent experiments. (D) Representative confocal micrograph of RAW 264.7 cells co-expressing the tandem PH domain of Lpd (green) and either BFP-INPP4B(C842A)-CAAX (left) or BFP-INPP4B-CAAX (right) during phagocytosis. Asterisks represent sites of phagocytosis; dotted line delineates the periphery of the cell. Similar results were observed in five independent experiments. ****, P < 0.0001.
