Figure 7

FRET-based "biosensing " by AKAR2-AKAP12 in response to beta-adrenergic agonist: effects of HT-31 peptide interference of AKAP/RII subunit-binding. Stably transfected A431 clones expressing AKAR-AKAP12 were serum starved over night, pre-treated with liposomes loaded with the HT-31 peptide (50 μM loading), and then stimulated with beta-adrenergic agonist (isoproterenol, 10 μM) for 10 min. Upper panel, images were recorded for CFP and YFP from the HT-31 peptide-treated cells that were then incubated either without isoproterenol (-ISO), or with isoproterenol (+ISO, 10 μM) for 10 min. The measurements were made in the perinuclear, cytoplasmic area (open red circle), an area in which robust FRET signals from activated AKAR2-AKAP12 are observed. Lower panel, the fluorescence density measurements were collected from a large array of images collected for this purpose. The fluorescence density of CFP in the control cells (-HT31) at basal state (-ISO) is highlighted by a red dashed line.