P4M localization is dependent on endosomes/lysosomal PtdIns4P. (A) COS-7 cells transfected with the PtdIns3P (PI3P) reporter GFP-FYVE-EEA1 and mCherry-P4M imaged by spinning-disk confocal microscopy during treatment with 100 nM wortmannin, a PtdIns 3-kinase (PI3K) inhibitor. (B) FRB targeted to late endosomes/lysosomes by fusion to Rab7 recruits lipid phosphatase PJ-Sac (active against PtdIns4P, PtdIns3P, and PtdIns(3,5)P₂) or MTM1 (active against PtdIns3P and PtdIns(3,5)P₂ but not PtdIns4P) in cells transfected with GFP-P4M. (C) FRB targeted to early endosomes by fusion to Rab5 recruits PJ-Sac or MTM1 in cells transfected with GFP-FYVE-EEA1. (D) FRB-Rab7 recruits a PIP5K or its catalytically inactive D253A mutant; the effect on PtdIns(4,5)P₂ generation is monitored with the PtdIns(4,5)P₂ reporter PH-PLCδ1-GFP. Graphs are grand means ± SEM from three independent experiments. Statistical significance (P < 0.05) is depicted over the indicated range (two-way ANOVA; see Materials and methods). n.s. = i.e., P > 0.05. rapa, rapamycin. Bars, 10 µm.