Skip to main content
. Author manuscript; available in PMC: 2014 Mar 28.
Published in final edited form as: Cell. 2013 Mar 28;153(1):216–227. doi: 10.1016/j.cell.2013.02.047

Figure 7.

Figure 7

Perinuclear PI4P is a substrate for mAKAP-scaffolded PLCε, stimulated by either Epac or ET-1 receptors. (A) NRVMs transfected with FAPP-PH-GFP were analyzed by live cell confocal microscopy before and after treatment with 10 μM cpTOME for 1 h. (See also Supplemental Movie 2) (B) Individual regions GFP fluorescence in NRVMs transfected with FAPP-PH-GFP were monitored with confocal microscopy and followed with time after treatment with vehicle, 10 μM cpTOME, 50 nM ET-1 or 50 nM ET-1+ 100 nM BQ-123 (top panels are representative traces). Data was pooled from 4 experiments at 0 and 50 min for quantitation and statistics (Bottom panels). (C) NRVMs were treated with Vehicle or 10 μM cpTOME for 50 min followed by extraction of PI4P and assay using a PI4P protein-lipid overlay assay according to the manufacturer's instructions. Data from three separate experiments are quantitated in the bottom panel and analyzed by a student's t-test. (D) NRVMs were cotransfected with FAPP-PH-GFP and either mAKAP-SR1 or control plasmid. Perinuclear GFP fluorescence was monitored as in B. ET-1 experiments in B and D were done in parallel so the ET-1 alone representative traces are the same in both panels. Top panel is a representative trace and bottom panel is pooled data from 3 experiments analyzed by one way ANOVA. (E) NRVMs were transfect with FAPP-PH-GFP and transduced with viruses expressing PLCε siRNA or random control siRNA and perinuclear GFP fluorescence was monitored as in B and D. Data are pooled from 5 independent experiments each. (F) NRVMs were transfected with YFP-C1b-Y123W to detect DAG localization. Left panel shows localization of YFP-C1b-Y123W by confocal microscopy. Right panel: NRVMs transfected with YFP-C1b-Y123W were stimulated with 10 μM cpTOME and perinuclear regions and cytoplasm were imaged over time. The ratio of perinuclear fluorescence (Fpn) to the cytoplasmic fluorescence (Fc) was calculated and normalized to the starting Fpn/Fc before cpTOME addition. Data are pooled from 4 independent experiments, Fpn/Fc at 9–12 min were individually compared with Fpn/Fc at 0 min with a one way ANOVA. G) Golgi specific depletion of PI4P blocks ET-1-dependent nuclear PKD activation. Left panel; cells were transduced with GFP-tagged, Golgi targeted Sac-1 and imaged by confocal microscopy. Right panel: Cells were cotransfected with plasmids expressing nDKAR and Golgi targeted Flag-tagged Sac-1 plasmids and nDKAR FRET was monitored as in Fig. 4 D and F. 50 nM ET-1 was added at the indicated time. Data pooled from 4 independent cells for each treatment from 2 separate NRVM preparations. All data are +/− SEM. See also Figures S6 and S7.