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Figure 8 | Journal of Molecular Signaling

Figure 8

From: “Shaping” of cell signaling via AKAP-tethered PDE4D: Probing with AKAR2-AKAP5 biosensor

Figure 8

Expression of the AKAP12 engineered with Δ1-145,T/P395 AKAP5 fragment which binds PDE effectively reverses PKA-catalyzed phosphorylation. HEK 293 cells were placed on glass bottom dishes. At 24 h later, cells were transfected with plasmids encoding either AKAR2-AKAP5 (A), AKAR2-AKAP12 (B) or AKAR2-AKAP12-[Δ1-145,T/P395] biosensors. At 48 h, cells were washed twice with DMEM with 4.5 g/L glucose and sodium pyruvate lacking L-glutamine and phenol red. At time = 0 cells were stimulated with 10 μM ISO and the FRET signal collected and monitored in real time. The ratios of yellow-to-cyan were recorded at every 5 s using METAFLUOR software and normalized by dividing all ratios by the emission ratio before stimulation. Experiments were repeated three times with equivalent results.

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