Figure 3

Continuous in vivo imaging of AKAP5 and AKAP12 phosphorylation by PKA following stimulation with β-adrenergic agonist, as reported by their AKAR2 biosensor derivatives. HEK 293 (A) and A431 cells (B) were transfected with the expression vectors for AKAP5 and AKAP12 biosensors, as well as the AKAR2 sensor itself. At 24 h post transfection, cells were washed twice with DMEM with 4.5 g/L glucose and sodium pyruvate lacking L-glutamine and phenol red. Cells were treated at time = 0 with 10 μM isoproterenol (ISO). The ratios of yellow-to-cyan signals were recorded every 5 s making use of METAFLUOR software. The data are normalized by dividing all ratios by the emission ratio captured in the basal sate before the stimulation. Experiments were repeated at least three times with equivalent results.