CREB phosphorylation and Rap1 pulldown analysis of B-CLL cells after treatment with nonselective or EPAC-selective agents. (A) CREB Ser133 phosphorylation in B-CLL cells treated for 30 minutes with media alone (CT), 10 μM rolipram (R), 40 μM forskolin (F), rolipram and forskolin (RF), 200 μM 8-Br-cAMP (Br), 200 μM N6-benzoyl-cAMP (Bz), 50 μM 8CPT-2Me-cAMP (CP), or 300 nM phorbol myristate acetate (PMA). (B) Same as for panel A, but where indicated B-CLL cells were pretreated for 10 minutes with the PKA-inhibitor H-89 (10 μM). (C) Rap1 activation following one hour of treatment with vehicle alone (CT), rolipram/forskolin (RF), 40 μM forskolin alone (F), 10 μM rolipram alone (R), 10 μM cilostamide (Cilo), or 10 μM IC242 was determined in leukemic cell preparations from 2 B-CLL patients using a GST-Ral-GDS pulldown technique, followed by anti-Rap1 immunoblotting (Pulldown). Cilostamide is a PDE3 inhibitor, whereas IC242 is a PDE7 inhibitor. As a control, total levels of Rap1 in whole cell lysates are shown (WCL). (D) Time course of Rap1 activation and CREB Ser133 phosphorylation following treatment of B-CLL cells with 10 μM rolipram. (E) Time course of Rap1 activation and CREB Ser133 phosphorylation following treatment of B-CLL cells with 50 μM 8CPT-2Me-cAMP. Control blots for total Rap1 and total CREB showed equal amounts of these proteins in the whole cell lysates used in panels D-E (data not shown).