PM targeting of PKCζ and Par-6 depends on both PI4P and PIP₂. (A) INPP5E converts PIP₂ to PI4P, which can be further converted to PI by Sac, whereas PJ converts both PIP₂ and PI4P to PI. Box: FKBP-PJ can be acutely recruited to PM through rapamycin (rapa)-induced heterodimerization with PM-anchored Lyn11-FRB. PM recruitment of PJ results in acute depletion of both PI4P and PIP₂. (B) PM localization of PKCζ::GFP and Par-6::iRFP was quantified before and after rapamycin addition in HEK293 cells expressing Lyn11-FRB-CFP and mCherry-FKBP-PJ, -Sac, -INPP5E, or -PJ-dead (as a negative control). Representative time-lapse images of Lyn11-FRB-CFP (red), PKCζ::GFP (green), Par-6::iRFP (magenta), and mCherry-FKBP-PJ/Sac/INPP5E/PJ-dead (cyan) are shown under each quantification figure. For each quantification, means ± SEM from 20–30 cells pooled across three independent experiments were plotted. (C) Purified GST-PKCζ/Par-6::His6 complex, but not purified GST-PKCζ alone or GST-PKCζ^KR8Q/Par-6::His6 complex, bound to PI4P- and PIP₂-liposomes. Scale bars: 5 µm (B, all panels).