Extended Data Fig. 6 |. Characterization of functional S_f-MOR, S_f-SecR, and S_f-mGluR2 compared to controls in living cells.

a, Dose-response curves for BRET-based cAMP inhibition and generation assays confirming S_f-MOR (top) and S_f-SecR (bottom) functionality, respectively. Curve fitting details are described in Extended Data Fig. 1b legend. b, Surface densities prior to smFRET imaging of donor and acceptor labeled samples for smFRET studies. Dots represent the number of acceptor (nAcc) or donor (nDon) particles per area for single cells. Box plot details are described in the legend of Extended Data Fig. 2b. The densities for S_f-mGluR2 are reproduced from Extended Data Fig. 2b for comparison. The median density of total labeled (acceptor + donor) TM proteins ranged from 0.28 – 0.36 molecules/μm². c, Distribution of smFRET events per cell for S_f-mGluR2 labeled with donor and acceptor (Don/Acc) (16 cells) compared to those for acceptor-only (16 cells) and donor-only (22 cells) controls as determined by the NLT analysis criteria. Dots represent the number of smFRET trajectories per area for each cell. Box plot details are described in the legend of Extended Data Fig. 2b. One-way ANOVA (DF = 53; F-value = 75.5) and Tukey post-hoc comparison were performed to obtain p-values (****p « 0.0001; n.s. = 0.996). The sum of the mean number of events per cell for the controls represent < 2% of those from Don/Acc S_f-mGluR2. d, Distribution of the duration of smFRET events of 2,695 smFRET trajectories for S_f-mGluR2 from 16 cells with the single-exponential with decay τ.