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Fig. 5 | BMC Biology

Fig. 5

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

Fig. 5

ScallOp2 and AmphiOp1 exhibit unusual signaling. a HEK293T cells were transfected with Glo22F, +/- ScallOp2, and treated with 2 μM forskolin for 30 mins prior to experiment to elevate basal cAMP, in an assay for opsin coupling to endogenous Gi. Cells were stimulated with 470 nm light. ScallOp2 acted to elevate cAMP in response to light in these conditions, therefore the maximum cAMP level post-flash was recorded as the response for each trial, for both + ScallOp2 and –ScallOp2 data sets. b HEK293T cells were transfected Glo22F, +/- ScallOp2, and GsX as indicated. They were treated with pertussis toxin (PTX) and stimulated with 470 nm light. Maximum cAMP post-flash was recorded. c,d HEK293T cells were transfected with Glo22F, +/- AmphiOp1, and GsX as indicated. They were treated with PTX and stimulated with 470 nm light as indicated. Time courses of AmphiOp1 response (+Gst) to different radiant exposures are shown in (c) (mean +/- SEM, n = 3). Because AmphiOp1 both elevated and suppressed cAMP levels after a light flash (depending on flash intensity), we recorded the largest deviation from baseline as the response for each trial. Average responses and SEM (n = 3) for each radiant exposure tested are shown in (d), with connecting lines for AmphiOp1 + GsX conditions. The symbols for –opsin/-GsX and + AmphiOp1/-GsX are offset slightly in the x-axis to aid visualization. Error bars smaller than symbols are not shown. AmphiOp1 amphioxus opsin 1, PTX pertussis toxin, ScallOp2 scallop opsin 2, SEM standard error of the mean

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