PM targeting of Lgl requires inositol phospholipids on the PM. (A) HEK293 cells expressing mLgl::GFP or Lyn11-FRB::CFP and RFP::FKBP12-PJ (PJ) to degrade PI4P and PIP2, RFP::FKBP12-PJ-Sac (PJ-Sac) to degrade PI4P, mRFP::FKBP12-INPP5E (INPP5E) to degrade PIP2, or FKBP12-PJ-dead::RFP (PJ-dead) as a control. The addition of rapamycin induced membrane recruitment of the FKBP12-tagged enzymes. Only combined depletion of PIP2 and PI4P causes complete diffusion of mLgl::GFP, whereas complete dissociation of iRFP::PLCδ_-PH and iRFP::SidM-P4M indicated complete depletion of PIP2 or PI4P by INPP5E and PJ-Sac, respectively. Graphs are means ± SEM of 11 ≤ n ≤ 19 cells from three independent experiments. (B) In Drosophila follicular cells expressing nuclear RFP, mRFP::FKBP-5Ptase (5Ptase) and Lck-FRB::CFP (not depicted), rapamycin addition induced PM recruitment of 5Ptase and dislocalization of Lgl::GFP from PM. Images were captured at 40 min after rapamycin or DMSO addition (n = 4 for each sample). (C) Liposome pull-down assays of GST and GST fusions of Lgl loop (Loop^WT), Loop^K6A, or Loop^K12A. **, P < 0.01; ***, P < 0.001. Bars, 5 µm. Error bars represent means ± SEM.