Figure 7

Probing the role of tethered PDE in AKAP5-based PKA-catalyzed phosphorylation. Wild-type HEK293 cells were untreated (Controls) or pretreated with siRNAs targeting PDE4D5, or stably transfected to express the Δ1-145,T/P395 AKAP fragment (a PDE-binding “sink”). These different cell treatments were followed by transfection with the AKAR2-AKAP5 biosensor. An addition batch of cells transfected with an expression vector harboring AKAR2-AKAP5 were pretreated with the PDE4-specific inhibitor Rolipram (10 μM for 10 min). The images display the YFP/CFP emission ratio images of HEK 293 cells derived from the AKAR2-AKAP5 biosensor in cells stimulated with ISO. The first images of each array are YFP-only images. Cells were treated with at time = 0 with 10 μM ISO and the imaging commenced. Representative pseudocolor images show the change in FRET in response to ISO (10 μM) stimulation at various time points recorded at every 5 s by METAFLUOR software.