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Fig. 4. FRAP analysis of GFP-GAP1^IP4BP in cells transfected with Ins(1,4,5)P₃-3-kinase. This figure illustrates a typical experiment to explore the effect of Ins(1,3,4,5)P₄ generation on the mobility of GAP1^IP4BP, with this particular example being from cells transfected with Ins(1,4,5)P₃-3-kinase in order to maximize Ins(1,3,4,5)P₄ production. (A) Subcellular distribution of GFP-GAP1^IP4BP and DsRed-Ins(1,4,5)P₃-3-kinase in co-transfected HEK-293 cells. The plasma-membrane distribution of GFP-GAP1^IP4BP (i) contrasts with the cytosolic distribution of the modified DsRed-Ins(1,4,5)P₃-3-kinase (ii); (iii) a merged image. (B) A 3 µm ROI was subjected to three photobleaches at the times indicated. Following the first photobleach (B1), carbachol (Cch) was added 5 minutes before the second photobleach (B2). Following fluorescence recovery of the second photobleach (B2), atropine (At) was added and, 2 minutes after At addition, the ROI was bleached for a third time (B3). This protocol was repeated in the absence of Cch and At, and in HEK-293 cells co-producing a catalytically dead DsRed-Ins(1,4,5)P₃-3-kinase variant or in cells transfected with only GAP1^IP4BP; the pooled data are presented in Table 2. Bar, 10 µm.

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