GFP-P4M reveals pools of PtdIns4P accessible to all of the lipid’s anabolic and catabolic enzymes. (A) COS-7 cells transfected with mCherry (mCh)-P4M and a fluorescent protein conjugate of each of the four human PI4K isoforms. Cells expressing mTq2-tagged PI4KA and mCherry-P4M were cotransfected with GFP-EFR3B and iRFP-TTC7B (insets) that together recruit the kinase to the PM. Colocalizing pixels are highlighted in the nMDP imaged. (B) Cells transfected with GFP-P4M and mCherry-tagged Sac proteins: either wild type (WT), the C389S catalytically inactive mutant, leucine-zipper alanine (LZA) ER export–deficient mutant, or K2A Golgi retrieval–deficient mutant. The graphs show the relative P4M intensity at the Golgi, PM, and Rab7-positive compartments (box and whiskers as in Fig. 7; boxes denote the interquartile range with lines at the median; whiskers denote the 10th and 90th percentiles). The numbers above each group refer to the p-value (one-way ANOVA, 30 cells per group; see Materials and methods) compared with the C389S mutant or as indicated. Bars, 10 µm.