INPP4B-mediated depletion of PtdIns(3,4)P ₂ disrupts phagocytosis. (A) Representative confocal extended-focus projections of doxycycline-inducible RAW264.7 macrophages expressing BFP-INPP4B(C842A)-CAAX (Control) or BFP-INPP4B-CAAX (PtdIns[3,4]P₂-depleted) following a 10 min incubation with IgG-opsonized SRBCs. Inside-outside staining was performed to differentiate between internalized/total (green) and non-internalized (red) SRBCs (see Materials and methods). (B) Quantification of the phagocytic efficiency in BFP-INPP4B(C842A)-CAAX and BFP-INPP4B-CAAX-expressing RAW 264.7 macrophages. Unpaired t-test of individual phagocytic efficiency values of n = 228 and 267 cells, respectively across N = 3 independent experiments; data are mean ± SEM. (C) Representative live confocal sections of RAW 264.7 macrophages expressing LifeAct-GFP and either BFP-INPP4B(C842A)-CAAX or BFP-INPP4B-CAAX during phagocytosis of IgG-opsonized SRBCs (red). Dotted boxes represent the phagocytic cups tracked in D. (D) Time-lapse microscopy of F-actin dynamics monitored by GFP-Lifeact during phagocytosis of IgG-opsonized SRBCs in RAW 264.7 macrophages expressing BFP-INPP4B-CAAX (top) or BFP-INPP4B(C842A)-CAAX (bottom; 0, 60, 120, and 180 s timepoints). Scale bar = 3 μm. Similar results were observed in 10 independent experimental repeats. ****, P < 0.0001.