Presence of raft markers in reticulocyte exosomes. (A) Equivalent protein amounts of ghosts, exosomes, and LDTI membranes from rat reticulocytes (exceeding 70%) were loaded on 10% SDS-PAGE and analyzed by Western blotting for Lyn and the TfR. (B) Because of GARP-50 antibody specificity, human red cells (6% reticulocytes) were subcultured in vitro. Red cell ghosts and exosomes collected from the medium were analyzed by Western blotting for human stomatin (GARP-50) and then for flotillin. (C) Rat reticulocytes (40 μL packed cell volume) were incubated with (50 μg/mL) biotinylated B-subunit of cholera toxin (bCTXB) for 3 hours at 4°C and chased for 60 minutes at 37°C. Cells were then fixed in 2% paraformaldehyde and processed as described in “Materials and methods.”