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Figure 4 | Journal of Molecular Signaling

Figure 4

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

Figure 4

Activation of AKAP5 in vivo : analysis of RII-binding site disruption and chemical inhibition of PKA. HEK 293 cells were grown on glass bottom dishes. At 24 h later, cells were transfected with expression vectors encoding either AKAR2-AKAP5 (A) or AKAR2-AKAP12 (B). At 48 h, cells were washed twice with DMEM with 4.5 g/L glucose and sodium pyruvate lacking L-glutamine and phenol red. Cells were treated with either the sterated Ht-31 peptide (50 μM), or sterated Ht-31 control peptide (50 μM) for 30 min in a humidified atmosphere of 5% CO2 and 95% air at 37°C. For FRET data, black lines trace the biosensor signal in non-treated cells; grey lines trace the signal from Ht-31control peptide-treated cells; light grey lines trace the signal from Ht-31 peptide-treated cells. Likewise, cells expressing AKAR2-AKAP5 cells were treated with PKA inhibitors, either H89 (10 μM, dots trace the FRET signal) or KT-5720 (1 μM, dashes trace the FRET signal) (B) for 10 min before ISO stimulation. Non-treated cells represent the control. At time = -25 s the monitoring of the FRET signal commenced; at time = 0, cells were stimulated with 10 μM ISO. Experiments were repeated three times with equivalent results.

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