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Fig. 6. PLD2 regulates activation of PIPkin I ${gamma}$ b. (A) The localisations of GFP-tagged PLD1 and 2 with FLAG-tagged PIPkin I ${gamma}$ were determined for cells in suspension (spun down on to a cover-slip). The data shown is that of PIPkin I ${gamma}$ b, but I ${gamma}$ a and I ${gamma}$ c gave identical results (supplementary material Fig. S5A). The inset image represents an image at a further 10x magnification. (B) Following transfection with GFP, GFP-tagged PLD2, FLAG-tagged PIPkin I ${gamma}$ bD316K and co-transfection of GFP-tagged PLD2 and FLAG-tagged PIPkin I ${gamma}$ bD316K adhesion to serum-coated plastic was quantified. Using a Student's t-test, a P values of <0.05% was obtained (*) between these transfectants. (C) Following maintenance in suspension for 60 minutes, the level of adhesion to serum-coated plastic was quantified for cells transfected with GFP-tagged PIPkin I ${gamma}$ b or the GFP-tagged PH domain from PLC ${delta}$ 1, in the presence of diC8-PtdOH or vehicle. The level of adhesion for each individual transfectant was obtained from an average of three experiments and error bars represent the standard deviation from the mean. Similar results were obtained when PLD-activity was suppressed in the presence of butan-1-ol (data not shown). (D) Following maintenance in suspension for 60 minutes, the localisation of GFP-tagged PLC ${delta}$ 1 was analysed in the presence of diC8-PtdOH or vehicle. Similar results were obtained when PLD-activity was suppressed in the presence of butan-1-ol (data not shown). All images are confocal and similar results were obtained in at least three separate experiments. Bar, 10 µm.

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