FIG. 6.
cAMP induces Rap1 activation. (A) Serum-starved HUVECs kept in medium 199 containing 1% BSA overnight were stimulated with cAMP-elevating agonists for 2.5 min as indicated at the top and at the concentrations described in the legend to Fig. 1A. GTP-bound Rap1 was detected by pull-down assay as described in Materials and Methods. Activation indicates the ratio of the poststimulation GTP-Rap1 intensity of total Rap1 intensity to the prestimulation GTP-Rap1 intensity of total Rap1 intensity. (B) Rap1 activation was analyzed by detecting GTP-bound Rap1 with lysates from HUVECs stimulated with PGI2 for 2.5 min at the different concentrations indicated at the top. (C) Rap1 activation was analyzed by detecting GTP-bound Rap1 with lysates from cells stimulated with 10-μg/ml PGI2 for the time period indicated at the top. (D) Serum-starved HUVECs similar to those described in the legend to panel A were stimulated with the reagents indicated at the top for 10 min at the same concentrations described in the legend to Fig. 1A. Rap1 activation was assessed by a method similar to that described for panel A. (E) The effect of 10 μM FSK on time-dependent Rap1 activity was examined as described for panel C. Representative results from at least three independent experiments are shown for all panels.