Fig. 4: DISCo-BRET competition binding experiments identify ligands binding to the two distinct FFA1 binding sites. | Nature Communications

Fig. 4: DISCo-BRET competition binding experiments identify ligands binding to the two distinct FFA1 binding sites.

From: Multicolored sequential resonance energy transfer for detection of simultaneous ligand binding at G protein-coupled receptors

Fig. 4

Competition DISCo-BRET binding experiments were conducted in the presence of both the red tracer TUG-2591 (100 nM) and the green tracer TUG-2597 (316 nM). Data are shown as the ratio of 690 nm / 540 nm luminescent emission (A), or 540 nm / 450 nm luminescent emission (B) and expressed as specific BRET above that obtained in the presence of both competing ligands, TUG-905 (10 μM) and T360 (10 μM). Competition binding data with various compounds are split over three graphs (i - iii), with the curve for TUG-905 shown as a dashed line in ii and iii for reference. All competition binding data are fit to a one site competition binding model, however given the complexity of allosteric interactions between ligands binding to site one and two, it is recognised that this model does not fully describe the binding reality. DISCo-BRET competition data are mean ± SEM from N = 10 (TUG-905), N = 7 (TUG-770 and T360), N = 5 (Cpd 6h), or N = 4 (all other compounds) independent experiments. NanoBRET competition binding experiments using the N terminal Nluc tagged FFA1 construct and either 100 nM TUG-2591 (Ci) or 316 nM TUG-2597 (Cii), or 2 μM TUG-2884 (D) are shown. Specific binding is shown after subtracting BRET obtained with 10 μM TUG-905 (Ci and D), or 10 μM T360 (Cii). NanoBRET data are mean ± SEM from N = 3 (Ci), N = 4 (Cii), or N = 7 (D) independent experiments. Source data are provided as a Source Data file.

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