Figure 5

FRET-based "biosensing" by AKAR2-AKAP12 in response to beta-adrenergic agonist: effects of addition of PKA inhibitor KT5720. Stably transfected A431 clones expressing AKAR-AKAP12 were serum starved over night and then stimulated with beta-adrenergic agonist (isoproterenol, 10 μM) for 30 min. Upper panel, images were recorded for CFP and YFP in the absence (0), following the 30 min challenge with isoproterenol (+ISO, 30 min), and in the presence of isoproterenol for 30 min, then treated with PKA inhibitor KT5720 (+ISO 30 min, +KT5720) The measurements were made in the perinuclear, cytoplasmic area (open red circle) 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 same area of the cells at basal state (-ISO) is highlighted by a red dashed line.