Fig. 2: BRET assay for assessing direct interactions between OMP and cAMP.

a Computer-simulated docking conformation between cAMP and OMP. b Schematic diagram of BRET between Rluc-OMP and 8NBD-cAMP and a competitive assay in addition to unlabelled cAMP. c Representative BRET-emission spectra in the presence of different concentrations of 8NBD-c AMP. d ΔBRET for Rluc-OMP^Wt. n = 8, 6, 25, 26, 23, 24 and 24 plates. e The decrease in ΔBRET (ΔΔBRET) for cAMP as a competitor. n = 3 plates. f Schematic localisation of mutated residues in Rluc-OMP. g Immunocytological confirmation of the cytosolic expression of the mutated Rluc-OMP fusion proteins. h Western blot confirmation of the sizes and concentrations of OMP and mutant Rluc-OMP fusion proteins. i ΔBRET for the various mutated Rluc-OMP proteins. Wt is the same as that shown in (d) for comparison. *P < 0.001. Experiments were repeated three times with similar results in (g, h). Statistics: (i) one-way ANOVA at different concentrations of 8NBD-cAMP with post hoc one-sided Dunnett’s multiple-comparison test for OMP^WT (n = 32) vs OMP^Mut: 0 μM, F(6, 96) = 0.0386, P_ANOVA > 0.99, P_Dunnett > 0.99 for all; 0.1 μM, F(6, 97) = 3.048, P_ANOVA = 0.00921, P_Dunnett < 0.01 for OMP¹²⁶ and OMP^126/136 and >0.1 for the others. In all, 0.2 μM, F(6, 97) = 7.539, P_ANOVA < 0.001, P_Dunnett < 0.01 for all; 0.5 μM, F(6, 105) = 16.05, P_ANOVA < 0.001, P_Dunnett = 0.0265 for OMP^90/126/136 and <0.01 for the others; 1 μM, F(6, 98) = 72.17, P_ANOVA < 0.001, P_Dunnett < 0.001 for all; 2 μM, F(6, 103) = 78.78, P_ANOVA < 0.001, P_Dunnett = 0.9131 for OMP¹²⁶ and <0.0001 for the others; 5 μM, F(6, 78) = 47.84, P_ANOVA < 0.001, P_Dunnett = 0.7257 for OMP¹²⁶ and <0.001 for the others; 10 μM, F(6, 87) = 47.41, P_ANOVA < 0.001, P_Dunnett = 0.0834 for OMP¹²⁶ and <0.001 for the others. n = 16, 11, 15, 8, 8 and 8 for OMP¹²⁶, OMP⁹⁰, OMP¹³⁶, OMP^126/90, OMP^126/136 and OMP^90/126/136, respectively. Mean ± s.e.m.