The GFP-PLCδ1 PH domain preferentially binds PIP₂ on the cytoplasmic face of the plasma membrane of loaded-erythrocyte ghosts. (A) Single optical sections of fluorescence micrographs (60×/1.42 oil objective [Olympus]) of erythrocyte ghosts loaded with recombinant GFP-PLCδ1 PH domain (5 μM) protein. Scale bar equals approximately 5 μm. (B) Membrane association of GFP-PLCδ1 PH domain in loaded ghosts. Ghosts loaded with GFP-PLCδ1 PH domain were lysed and fractionated into membrane and cytoplasmic fractions by centrifugation, separated by SDS-PAGE, and analyzed for recombinant protein by anti-GFP immunoblotting. T, total PLCδ1 PH domain–loaded ghost lysate; C, cytoplasmic fraction; M, membrane fraction; 5 × 10⁷ cells per lane; protein loading equal by Ponceau staining. Purified GFP-PLCδ1 standard is shown at right; nanogram scale. (C) Protease protection assay of GFP-PLCδ1 PH domain in loaded ghosts. Ghosts loaded with GFP-PLCδ1 PH domain were incubated in buffer with or without proteinase K and/or 1% Triton X-100 detergent, then separated and analyzed as in panel B. In addition to anti-GFP immunoblotting, control antibodies to erythrocyte flotillin-1 (flot1; completely protease protected) were also used. C, control; P, proteinase K; T, Triton X-100; P/T, proteinase K and Triton X-100; 2 × 10⁷ cells per lane; protein loading equal by Ponceau staining. (D) In vitro liposome association of GFP PLCδ1 PH domains. Recombinant GFP PLCδ1 PH domain fusion protein was incubated with purified liposomes containing PIP₂, PI4P, or PA, separated into a liposome-containing pellet (P) and liposome-depleted supernatant (S) fractions by ultracentrifugation, and immunoblotted for GFP as described in the other panels. Numbers represent the relative ratios of DMPC to PIP₂, PI4P, or PA in liposomes (0.1 = 1:20; 0.5 = 1:40; 0.025 = 1:80). *Lane with most PH domain binding of PIP₂-containing liposomes.