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