Figure 6

Activation of AKAP5 is reversed by tethered PDE. Wild-type HEK cells or cells stably transfected with Δ1-145,T/P395, an N-terminally truncated AKAP5 (lacking the PCDs that function in membrane localization) with a T/P395 mutation, were co-transfected with AKAR2-AKAP5 biosensor (A). At 24 h post biosensor transfection, cells were treated at time = 0 with 10 μM ISO (A,B,C). HEK293 cells were transiently transfected with AKAR2-AKAP5 and pretreated with either vehicle alone (DMSO) or 10 μM Rolipram (a chemical inhibitor of PDE4) for 10 min. These cells were treated with 10 μM ISO at time = 0 (B). Wild-type HEK293 and cells in which PDE4D5 was knocked down both were transiently transfected with AKAR2-AKAP5 biosensor. Cells were treated with 10 μM ISO at time = 0 (C). 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.