Fig. 3
FLIM–FRET results. HEK293 cells were transiently transfected with Gα-mCitrine alone or both D2R-mCherry and Gα-mCitrine (donor and acceptor) with or without Gβ1γ2, or the donor in the presence of the acceptor after treatment with 1 μM rotigotine; mCitrine lifetime was measured: a Gαi1-mCitrine; b Gαi2-mCitrine; c Gαi3-mCitrine; d Gαs-mCitrine. Fluorescence lifetimes are presented in a continuous pseudo-color scale representing the time values ranging from 2.7 (blue) to 3.7 ns (red). e–h Box-and-whisker plots of the fluorescence lifetime τ1 of energy donor (Gα-mCitrine) and donor in the presence of acceptor (D2R-mCherry) are provided. The median is shown as a line in the box, while the bottom and top boundaries represent the lower and upper quartile, respectively. Statistical significance of the difference in the fluorescence lifetimes of the donor (τ1) was detected in the absence and presence of the energy acceptor using Mann–Whitney U test (**p < 0.005, ***p < 0.0001). Gαi1: n = 58; Gαi1 and Gβ1γ2: n = 40; Gαi1 and D2R with rotigotine: n = 34, without rotigotine: n = 61; Gαi1 and Gβ1γ2 and D2R with rotigotine: n = 26, without rotigotine: n = 58; Gαi2: n = 49; Gαi2 and Gβ1γ2: n = 44; Gαi2 and D2R with rotigotine: n = 41, without rotigotine: n = 47; Gαi2 and Gβ1γ2 and D2R with rotigotine: n = 31, without rotigotine: n = 46; Gαi3: n = 50; Gαi3 and Gβ1γ2: n = 54; Gαi3 and D2R with rotigotine: n = 33, without rotigotine: n = 68; Gαi3 and Gβ1γ2 and D2R with rotigotine: n = 33, without rotigotine: n = 79; Gαs: n = 39; Gαs and Gβ1γ2: n = 39; Gαs and D2R: n = 55; Gαs and Gβ1γ2 and D2R: n = 48