Figure 7.

Calcium response promoted by β₁AR or β₂AR activation. (a) Schematic representation of the luminescence Obelin biosensor used to detect calcium. (b) Schematic representation of the mode of action of p115-RGS-CAAX (p115-CAAX) construct as a dominant negative construct for the inhibition of G_12/13 signalling. HEK 293 cells stably expressing HA-β₁AR were transfected with Obelin. (c) Kinetics of calcium response by indicated ligands (representative, isoproterenol, n = 10, other ligands n = 3) and expressed as relative luminescence units. (d) Concentration-responses curves for Ca²⁺ mobilization following β₁AR activation by indicated ligands. Data were normalized to maximal isoproterenol response (100%), and expressed as mean ± SEM values. Detail of the number of experiments, maximal responses, pEC₅₀ values and statistical comparisons of curve parameters for different ligands are provided in Supplementary Tables S1 and S2. (e) HEK 293 cells were transiently transfected with β₁AR or β₂AR along with the obelin biosensor, with or without the p115-CAAX inhibitor. Data were normalized to maximal A23187 response (100%), determined from the area under the curve (AUC), and are expressed as the mean ± SEM values (n = 3). Statistical comparisons were done using two-way ANOVA followed by post-hoc comparison with Tukey’s test. (f) Parental HEK 293 or ΔGαs cells were transfected with β₁AR or β₂AR, along with obelin, with or without Gα_s. Data normalization and statistical analysis were done as described in (e) (n = 3).