Fig 1.

Quantification of PKA dynamics using AKAR3. Images in part A show that upon treatment with forskolin (10 μM), fluorescence in the CFP channel decreased while fluorescence in the FRET channel increased. The ratiometric image shows the consequent increase in FRET/CFP emissions after treatment with forskolin. Graph B shows the quantification of CFP and FRET emissions over time. FRET intensity was corrected for CFP crossover (44%) at each time point. In graph C, the normalized ratio of FRET/CFP intensity was plotted over time, and the fold increase was calculated by dividing the maximal ratio value after treatment (R_max) by the mean ratio value prior to treatment (R₀). Graph D represents the mean fold increase in each of the AKAR3 constructs after forskolin treatment. Scale bar = 10 μm; Cells were incubated in SES with 10 μM IBMX for 30 minutes prior to forskolin treatment. The diagram in E [adapted from ref. 54] illustrates the principle underlying how the FRET biosensor works. PKA dependent phosphorylation of the substrate peptide results in binding of the peptide to a phosphoamino acid binding site on the FHA1 region of the biosenor and a change in the positioning of the fluorescent proteins relative to each other. Graph F illustrates the effect of different molar ratios of AKAR3:gravin plasmid DNA in the transfection mixture on the percentatge coexpression of the gravin construct in cells expressing AKAR3.