(A) HEK293 cells co-expressing FLAG-tagged β2AR, HA-tagged Gsα and EGFP were stimulated with 100 nM ISO or indicated β-blockers for 5 min. In proximity ligation assay (PLA), cells were immuno-stained with HA and β2AR antibody, nuclei were counterstained with DAPI. The green EGFP signal represents transfected cells, and red PLA signal represents Gsα and β2AR interactions. Carvedilol and alprenolol promoted Gsα recruitment to β2AR, but timolol could not. Scale bar, 10 μm. Representative of n = 15, 16, 16, 17, 18 and 18 images respectively, three experiments. (B–C) HEK293 cells expressing ICUE3 biosensor were treated with 1 μM ISO or indicated β-blockers (B), or together with 100 μM phosphodiesterase inhibitor IBMX (C). (D–E) HEK293 cells expressing β2AR-ICUE3 biosensor were treated with indicated concentration of ISO or β-blockers. In some cases, cells were pretreated for 30 min with the β2AR antagonist ICI (10 μM) or the adenylate cyclase inhibitor ddA (50 μM) before adding β-blockers. Changes in ICUE3 FRET ratio (an indication of cAMP activity) were measured. Experiments were performed in the presence of 1 μM β1AR-selective antagonist CGP20712A to block endogenous β1AR signaling. Mock, no primary antibody; NT, no treatment; ISO, isoproterenol; TIM, timolol; ALP, alprenolol; CAR, carvedilol; ICI, ICI118551; PRO, propranolol; MET, metoprolol; 177, CGP12177, IBMX, 3-isobutyl-1-methylxanthine; ddA, 2',5'-dideoxyadenosine. Each dot in the scatter dot plot in B–E represents a value from an individual tested cell. Error bars denote s.e.m., P values are computed by one-way ANOVA followed by Tukey’s test between NT (A) or TIM (B–E) and other groups.