Response to 8CPT-2-Me is not dependent on Epac or Rap1 protein levels in SS RBCs. (A) Response to 8CPT-2-Me is not dependent on the levels of Epac protein. An equivalent concentration of RBC lysate was derived from 9 previously characterized donors relative to 8CPT-2-Me-induced RBC adhesion to laminin (3 SS responders, 3 SS nonresponders, and 3 AAs). The lysate was separated on a 6% SDS polyacrylamide gel under reducing conditions, transferred to a PVDF membrane, and blotted for Epac1 with an Epac1 monoclonal antibody. PP2A was used as a loading control. Relative Epac protein levels were determined by taking a ratio of the densitometry value obtained from the Epac sample to the densitometry value from the corresponding PP2A loading control and plotted for each patient. (B) Response to 8CPT-2-Me is independent of basal GTP-Rap1 levels. RBC lysates from 11 patients (3 SS responders, 3 SS nonresponders, and 5 AAs) of equivalent RBC concentration were assayed for GTP-Rap1 with GST-RalGDS-RBD beads. Rap1 was then detected on a Western blot with a Rap1-specific antibody, with PP2A as a loading control. Relative Rap1 values were obtained by densitometry as described in Figure 2A. The relative GTP-Rap1 value obtained for each patient is represented as a dot.