PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 are differentially distributed within the phagocytic cup, and SHIP1 and SHIP2 are dispensable for PtdIns(3,4)P 2 production at this site. (A) RAW 264.7 macrophages co-expressing the cPHx2 (green) and PH-BTKx2 (magenta) constructs during phagocytic cup formation. (B) Intensity profile from the dotted line in A of the cPHx2 (green) and PH-BTKx2 (magenta) constructs at the phagocytic cup. Similar results were observed in three independent experimental repeats. (C) Representative confocal sections of RAW 264.7 macrophages co-expressing the cPHx3 probe (red) with either the wild-type GFP-SHIP2 (left) or the catalytically inactive GFP-SHIP2(D607A) (right). (D) Normalized phagosomal-cPHx3 fluorescence intensity in cells co-expressing either wild-type GFP-SHIP2 (n = 7) or the catalytically inactive GFP-SHIP2(D607A) (n = 11). Unpaired t test of individual values across n = 3 independent experiments; data are means ± SD. (E) Representative confocal slices of RAW 264.7 macrophages expressing the cPHx2 probe (green) in either control cells (left), or cells pre-treated with the SHIP2 inhibitor AS1949490 10 μM for 5 h (middle) or pre-treated with the pan-SHIP1/2 inhibitor K118 5 μM for 5 h (right). Asterisks indicate sites of phagocytic cup formation.
