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Figure 3


Fig. 3. The Gi/o protein anchor construct FRET vector is used to characterize G{alpha}i/o and G{alpha}i/obeta{gamma} microlocalizations. (A) Schematic representation of heterotrimeric Gi/o protein anchor constructs. M, myristoyl; P, palmitoyl; G, geranylgeranyl; FP, fluorescent protein (i.e. either mCFP or mCit). Source refers to the proteins from which targeting sequences were derived. (B) The subcellular localization of Gi/o-anchor constructs was imaged by confocal microscopy. Bar, 10 µm. (C) The FRET vectors show the Emax values of pairs of Gi/o anchor constructs and microdomain markers. Column headings give names of G-protein-anchor constructs, row headings give co-expressed microdomain markers. The Emax values are given in % ± s.d.; n, number of independent experiments. The value for Ni2C{gamma}-mCit/mCFP-tH is significantly larger than the G{alpha}i/o-anchor construct/FP-tH pairs (P<0.01, 2-tailed Student's t-test), whereas the value for Ni2C{gamma}-mCit/mCFP-tR is significantly smaller than that for the G{alpha}i/o-anchor construct/FP-tR pairs (P<0.001, 2-tailed Student's t-test). (D) Plots of the FRET efficiency (E) against the normalized acceptor surface concentration (cA) at a constant donor mole fraction of indicated FRET pairs with fitted curves as described (from left to right, {chi}2: 3.1 and 6.2).





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