Rap1 is activated by the cAMP pathway in SS RBCs. (A) Forskolin stimulates Rap1 in SS RBCs. Cells were pretreated with 200 μM IBMX for 30 minutes to inhibit phosphodiesterase activity. The cells were subsequently treated with 80 μM forskolin for the time points shown. After lysis, GTP-bound Rap1 was precipitated with GST-RalGDS-RBD beads and detected with a Rap1-specific antibody by Western blotting. The numbers shown below the blot are relative densitometry values obtained by taking a ratio of the densitometry value obtained from the Rap1 band to its corresponding PP2A loading control band. Values were then normalized to the zero time point. Densitometry for panels B and C was also analyzed in this manner. The 1-hour time point is from the same Western blot as the other time points. Data are representative of experiments from 3 different patient samples. (B) Dibutyryl (db) cAMP activates Rap1. SS RBCs were treated with 200 μM db cAMP for the indicated time points. GTP-bound Rap1 was detected as in panel A. Data are representative of experiments from 5 different patient samples. (C) Rap1 is activated by isoproterenol in SS RBCs. Cells were treated with 100 μM isoproterenol for the time points indicated. After lysis, GTP-bound Rap1 was detected as in panel A. Data are representative of experiments from 5 different patient samples.