PPIn production within the PM depends on PI delivery from the ER. (A–C and E–G) A schematic depicting the experimental design is provided above confocal images of representative cells showing the recruitment of FKBP–BcPI-PLC^AA R163A to the PM (B and F, left panels, green) or to the ER (C and G, left panels, magenta) after a 5-min treatment with rapamycin (100 nM). The localization of PI4P (B and C; EGFP-P4M_SidMx2) and PI(4,5)P₂ (F and G; PLCδ1_PH-EGFP) are provided before (center panels) and 20 min after (right panels) recruitment of FKBP–BcPI-PLC^AA R163A to the respective membrane compartments (scale bars, 10 μm). (D and H) Kinetics of PI4P (D) or PI(4,5)P₂ (H) levels within the cytosolic leaflet of the PM as measured using the PM-PI4P^BRET or PM-PI(4,5)P₂^BRET biosensors, respectively, in response to recruitment of FKBP–BcPI-PLC^AA R163A either directly to the PM (PM-FRB; green traces) or to the ER (FRB-ER; magenta traces). Treatment with GSK-A1 (100 nM; D, grey trace), which selectively inhibits PI4KA, or AngII (100 nM; H, grey trace), which stimulates PI(4,5)P₂ hydrolysis, are included as positive controls for the PM-PI4P^BRET and PM-PI(4,5)P₂^BRET biosensors, respectively, as well as to provide scale for any changes to PPIn levels that are associated with the membrane recruitment of FKBP–BcPI-PLC^AA R163A. BRET measurements are presented as mean values ± SEM from three independent experiments performed using triplicate wells.