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Figure 6


Fig. 6. FRET experiments with full-length heterotrimeric-G-protein constructs confirm displacement of the G{alpha} subunit into another microdomain after activation, as predicted by the FRET vectors of the anchor constructs. (A) Schematic representation of fluorescent full-length heterotrimeric-G-protein constructs. We fused the fluorescent protein (FP) to the N-terminus of the G{alpha} subunits and targeted these fusion constructs using the targeting sequences of the G-protein-anchor constructs. (B) Confocal imaging confirmed that all full-length G{alpha} constructs are predominantly localized to the plasma membrane. Bars, 10 µm. (C) FRET changes were calculated after stimulating cells with AlF4- (30 µM, 40 minutes, 22°C). Fluorescence of cells co-expressing indicated full-length G-protein constructs, microdomain markers and in addition unlabelled Gbeta1{gamma}2, was measured in a sensitive spectrofluorometer. Numbers at the bars give the average FRET change in percent with standard deviations and number of independent experiments, n, in brackets.





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