Fig. 2.

BRET experimental controls. (A) The maximum BRET signal was obtained when HEK 293T cells were transfected with KOR, Gβγ-Venus, and masGRK3ct-nLuc. Since there were no exogenous Gα subunits expressed, the Gβγ-Venus couples to masGRK3ct-nLuc, and a BRET ratio of 0.58 ± 0.037 was obtained. When 10 µM U50,488 was applied, there was no change in the BRET signal, signifying that the endogenous Gα proteins did not affect the signal. (B) The minimum BRET signal was obtained when Gβγ-Venus, masGRK3ct-nLuc, and a Gα subunit of interest were expressed. In this scenario, Gβγ-Venus coupled to the Gα subunit. Since HEK 293T cells did not endogenously express the KOR, when U50,488 was applied, there was no change from the baseline BRET signal. (C) Optimal assay conditions were obtained when KOR, Gβγ-Venus, masGRK3ct-nLuc, and Gα were expressed. In the baseline condition, Gβγ-Venus coupled to Gα, and thus there was a minimal BRET signal. When the agonist U50,488 was applied, the BRET ratio significantly increased (P = 0.003). Lastly, when the KOR antagonist nor-BNI was applied, the BRET ratio did not change from the baseline condition (P = 0.8). (D) Since KOR couples to the Gαi/o class of proteins, when Gαs was expressed with KOR, Gβγ-Venus, and masGRK3ct-nLuc, no signal was transmitted when 10 µM U50,488 was applied. Data are the mean BRET ratio from three independent experiments performed in duplicate ± S.D. (E) Baseline and U50,488-stimulated ratios for all experiments performed across the various Gα subunits. No statistically significant differences were observed between baseline and 10 µM U50,488–stimulated ratios between the Gα subunits. Data are mean BRET ratios ± S.D.