(A–F) PC12 cell membrane sheets were incubated with 2 μM PLCδ–PH-GFP fusion protein and 100 μM FM4-64 to image PI 4,5-P (A and B) or membrane area (C and D), respectively

(E and F) Membrane sheets were incubated with PLCδ–PH-GFP fusion protein after incubations with or without 2 mM MgATP, respectively. (G–I) Quantification of membrane PI 4,5-P. (G) Supported planar bilayers containing indicated mole percentage of PI 4,5-P and 0.1 mol% rhodamine-PE were incubated with 2 μM PLCδ–PH-GFP fusion protein and imaged in red (top) and green (bottom) channels. (H) Fluorescence intensity per pixel was plotted for each supported bilayer to generate a calibration curve. (I) From calibration curves prepared in parallel, pixel intensity on PC12 cell membrane sheets (incubated with or without MgATP) was used to infer PI 4,5-P concentrations in microdomains, between microdomains, and averaged across the membrane. Mean ± SEM values are shown for +MgATP ( = 13) and −MgATP ( = 5). (J and K) PI 4,5-P microdomains overlap with clusters of CAPS and docked vesicles. (J) PI 4,5-P microdomains in PC12 cell membrane sheets were localized by incubation with PLCδ–PH-GFP and fixed for immunolocalization with CAPS polyclonal or chromogranin B monoclonal antibodies. (K) Triple channel colocalizations were quantified as described in Materials and methods. Mean ± SEM ( = 9) values are shown.<p><b>Copyright information:</b></p><p>Taken from "Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion"</p><p></p><p>The Journal of Cell Biology 2008;182(2):355-366.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483516.</p><p>