Epac contributes to Rap1 activation in SS RBCs. (A) Epac is present in SS RBCs. SS RBC lysate or HEK293/Epac lysate was separated by SDS-PAGE. The proteins were then transferred to PVDF membrane and Western blotted with a rabbit anti-human Epac1 antibody (left and bottom) or a rabbit control antibody (right). Duplicate lanes are shown. (B) Treatment with an Epac-specific cAMP analog stimulates Rap1. SS RBCs were treated with 100 μM 8CPT-2-Me at the indicated time points. The cells were lysed, and GTP-bound Rap1 was detected as in Figure 1A. Densitometry values were calculated as described in Figure 2A. Results are expressed as mean ± SE from 4 separate experiments. (C) Stimulation of Rap1 via Epac promotes cellular adhesion to laminin. SS RBCs were treated with 100 μM 8CPT-2-Me at the times indicated. While still in the presence of 100 μM 8CPT-2-Me, the cells were then flowed over chambers coated with 0.75 μg laminin in a flow adhesion assay. Adhesion was quantified as described in “Patients, materials, and methods.” Results are expressed as mean ± SE from 1 of 3 similar experiments. (D) Inhibition of PKA has no effect on 8CPT-2-Me-stimulated adhesion. SS RBCs were untreated or pretreated with 87 nM PKAI for 1 hour and then 100 μM 8CPT-2-Me was added in with the PKAI for 20 minutes. The cells, while still in the presence of these pharmacologic agents, were flowed across chambers coated with 0.75 μg laminin in a flow adhesion assay. Adhesion was quantified as described in “Patients, materials, and methods.” Results are expressed as mean ± SE from 4 separate experiments.