Fig. 4

FLIM-FRET results. A Fluorescence lifetimes for the donor (Gαi₃-Citrine) and the donor in the presence of the acceptor (D₂R-mCherry). Data were collected from at least five independent experiments (n = 5) and are presented as mean ± 95% confidence interval (CI). The mean fluorescence lifetime of donor Gαi₃-Citrine was compared with the donor fluorescence lifetime in the presence of the acceptor D₂R-mCherry with an unpaired t-test (**** – p < 0.001). Because of the varying number of repeats, the median donor lifetime in the Gαi₃βγ setup was compared to donor–acceptor systems with different Gβγ dimers using the Mann–Whitney U test (**** – p < 0.001). B Representative fluorescence lifetime images for one of the FRET donor (Gαi₃-Citrine β₁γ₈; B1) and FRET donor–acceptor pair (Gαi₃-Citrine β₁γ₈ – D₂R-mCherry; B2). C Energy transfer efficiency (% E) between investigated Gαi₃-Citrine – D₂R–mCherry pairs; error estimated with exact differential