Polyanionic lipids do not affect the association of PILS-Nir1 with the PM, but do affect NES-PABDx2-Spo20 membrane binding. FKBP-tagged PIP phosphatases were recruited to the PM by 1 µM rapamycin (Rapa) inducing dimerization between the FKBP fragment and PM-localized FRB. Quantification of the recruitment of the FKBP-tagged constructs is shown in orange. The resulting depletion of PIPs is shown by the Tubby(C) biosensor for PIP₂ or the P4Mx1 biosensor for PI4P in gray. (A) FKBP-PJ-Dead does not affect PIP₂ or PI4P levels. (B) FKBP-PJ depletes both PIP₂ and PI4P at the PM. (C) FKBP-INPP5E depletes PIP₂ at the PM but does not reduce PI4P levels. (D) FKBP-PJ-Sac1 only depletes PI4P at the PM. Association of PILS-Nir1 (teal) or NES-PABDx2-Spo20 (pink) with the PM during recruitment of these phosphatases was determined using TIRF microscopy to analyze the fluorescence intensity at the basal membrane of the cells. The fluorescence at a given time (F_t) was divided by the fluorescence before rapamycin stimulation (F_pre). All xy graphs show the grand means of three to five experiments ± SEM. Total cells analyzed = 7–16. Representative TIRF images of the PILS-Nir1 and NES-PABDx2-Spo20 biosensors during rapamycin recruitment of the PIP phosphatases are shown to the right.