Figure 3. PI3K dependency of RhoGAP recruitment to phagocytic cups.

(a–d) RAW 264.7 macrophages were co-transfected with constructs encoding each of the 10 RhoGAPs that most markedly accumulated at phagocytic cups as well as with the plasmalemmal marker PM–RFP. Transfectants were exposed to IgG-opsonized erythrocytes (IgG–E; shown in blue) for 3 min before being fixed and imaged by confocal microscopy. Where indicated, cells were treated with either vehicle (DMSO) or the PI3K inhibitor LY294002 for 10 min before the addition of phagocytic targets. Only a subset of the 10 RhoGAPs that were investigated is shown, namely two PI3K-independent (a,b) and two PI3K-dependent (c,d) proteins. (see Supplementary Fig. 3 for the remaining RhoGAPs that were investigated.) Arrows point to sites of particle engagement. Scale bar, 10 μm. Micrographs are representative of three independent experiments. At least 20 cells were assessed per replicate for each of the RhoGAPs examined.