Rap1 is present in a pure fraction of red blood cells. (A) WBC contamination does not contribute to the observed Rap1 signal. RBCs were counted with a Coulter cell counter and diluted to 1 × 109 cells/mL. WBCs were counted microscopically on a hemacytometer and adjusted to 2 × 107 cells/mL (representing a 2% contamination level). The cells were lysed and subjected to a GST-RalGDS-RBD pull-down assay. Rap1 was detected by Western blotting with a Rap1-specific antibody. (B) Platelet contamination is not detectable in the RBC preparation. RBC and platelet lysates were probed by Western blotting with an αIIb-specific antibody as described in “Patients, materials, and methods.” (C) Both Rap1a and Rap1b are present in RBCs. Shown are MS/MS spectra corresponding to peptides from Rap1a and Rap1b obtained from tryptic digestion of Rap protein from a GST-RalGDS-RBD pull-down of RBCs.
