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. 2017 Dec 5;113(11):2396–2405. doi: 10.1016/j.bpj.2017.09.018

Figure 2.

Figure 2

Effect of receptor-derived pYp and H-Ras on net PI3Kα kinase activity and PIP3 production. (A) TIRFM images of pYp-activated PI3Kα lipid kinase activity on a target bilayer surface. Shown is the accumulation of product PIP3 lipid detected by the fluor-labeled PIP3 sensor (GRP PH domain), with and without membrane-anchored H-Ras. Each image is a square section (121 pixels or 29.0 μm per side) of the monitored TIRFM area (256 pixels or 61.4 μm per side). (B) Time course plotting the increasing number of PIP3 molecules per field under four activating conditions, illustrating strong synergy between the pYp peptide and H-Ras in activating PI3Kα kinase and PIP3 production. (C) The average slopes of these time courses, yielding average net rates of PIP3 production. Each average rate was generated from three replicates on each of nine or more different days (n = 27–36). In all cases in (A)–(C), H-Ras is loaded with non-hydrolyzable GTP analog GMPPNP.