Additional file 2: Figure S1. of A live cell assay of GPCR coupling allows identification of optogenetic tools for controlling Go and Gi signaling

Pertussis toxin, GsX point mutations, and comparison to JellyOp. a–c HEK293T cells transfected with Glo22F and rod opsin were treated with or without pertussis toxin (PTX) as indicated. Forskolin was added to elevate cAMP after 5 mins, and cells were flashed with light at varying intensities at 33 mins. The signal for each trial was normalized to pre-flash and the minimum cAMP post-flash was recorded for each trial. a,b Time courses of the GloSensor cAMP signal (average of three trials +/- SEM) for dark controls and 1015.1 photons/mm2 flash conditions. a Raw luminescence and b normalized to the final point prior to flash. Average responses +/-SEM for all light levels tested are shown in (c), with the best-fitting curve for –PTX. d HEK293T cells transfected with Glo22F, rod opsin, and Gso, Gsi, or Gst bearing the native Cys residue in the C-terminal -4 position were not treated with PTX. Cells were stimulated with light and the responses analyzed as in Fig. 3. Best-fitting maximum response amplitudes are graphed alongside the response amplitudes for rod opsin tested with PTX-insensitive Gso, Gsi, or Gst and treated with PTX (data reproduced from Fig. 3d). The wild-type and Ser point mutants for each GsX were compared by ANOVA. Uncorrected p values are shown and no differences were statistically significant. e HEK293T cells were transfected with Glo22F and either JellyOp or rod opsin, with or without exogenous G protein as indicated, and treated with PTX. Cells were stimulated with light and responses analyzed as in Fig. 3. The graph shows mean responses (n = 3, +/-SEM) at each light intensity. Lines are best-fitting sigmoid curves. Error bars smaller than symbols are not shown. (PDF 441 kb)