Figure 9

Probing the role of PDE in AKAP5–mediated signaling to ERK1,2. Wild-type HEK293 cells, cells treated with siRNAs targeting either AKAP5, AKAP12 or PDE4D5 or HEK293 cells stably transfected with an expression vector harboring Δ1-145,T/P395 AKAP5 fragment, or wild-type cells pretreated with the PDE4-specific inhibitor Rolipram (10 μM for 10 min) were stimulated with 10 μM ISO for different time points. Following the 10 min time course the cells were lysed and the lysates analyzed by SDS-PAGE. The resolved proteins were blotted and probed with activation- and phospho-specific antibodies to pERK1,2. Blots also were probed with antibodies to ERK1,2 (A). Expression of proteins targeted with siRNAs was established by immunoblotting of the same blots (B). Quantification of ERK1,2 activation in the three control and experimental conditions is displayed (C). The results shown are the activity ratio compared with the control and are the mean values and s.e.m. of three or more independent experiments. *, denotes significance of p < 0.05 of a ratio compare to AKAP5 deficient (AKAP5 KD) samples.