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Fig. 3 | BMC Biology

Fig. 3

From: A live cell assay of GPCR coupling allows identification of optogenetic tools for controlling Go and Gi signaling

Fig. 3

Human rod opsin, cone opsins, and rod opsin loop-3 chimeras couple to GsX. a Schematic of Gi/o/t-coupled opsin signaling through endogenous pathways (left) and redirected via Gsi, Gso, or Gst. Opsin is shown with all-trans-retinal, representing the active state of human visual opsins. be HEK293T cells treated with pertussis toxin and transfected with Glo22F cAMP reporter, opsin and Gso, Gsi, or Gst were stimulated with 470 nm light (rod opsin n = 3, others n = 4). GloSensor cAMP luminescence was normalized to baseline and light responses were calculated as detailed in “Methods.” b Time-course data for 1014.1 photons/mm2 flash at t = 0, showing mean +/- SEM at each timepoint. c Dose–response curves for each opsin/GsX combination, showing mean GloSensor cAMP response +/- SEM for each radiant exposure level tested. Curves are fitted to the average GloSensor response across replicates. The best-fitting parameters are listed in Additional file 7. d Individual trial response amplitudes from sigmoid curves for each replicate. Within each GsX, response amplitudes for the opsins were compared to that of rod opsin by ANOVA. e Individual trial response amplitudes in (d) were normalized to the average response amplitude for rod opsin for the appropriate GsX, to allow comparison of relative selectivity by ANOVA. For the ANOVA analysis in (d) and (e), α = 0.0033, based on a Bonferroni correction for multiple comparisons (initial α = 0.05, 15 comparisons total). Asterisks (*) represent comparisons that pass this threshold. Some error bars are smaller than the symbols, and in these cases the error bars are not shown. Best-fitting parameters and statistical tests pertaining to individual trials in (d) and (e) are in Additional file 10. LWS long-wave sensitive, RHO human rod opsin, RL3Am RHO loop 3/AmphiOp1 loop 3 chimera, RL3m6L2 RHO loop 3/mGluR6 loop 2 chimera, RL3Sc RHO loop 3/ScallOp2 loop 3 chimera, SEM standard error of the mean, SWS short-wave sensitive

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