Figure 5.

Ghosts remain permeable after lysis and Fl-E5-TAT remains tightly bound to their membranes. The fluorescent marker 10 kDa Dextran-FITC was added to cells lysed by treatment with E5-TAT (3 µM) at pH 4.5. A clear fluorescence contrast is observed between the exterior and interior of intact RBCs. Conversely, ghost display a fluorescence signal similar to that of the media surrounding them, suggesting that the fluorescent dextran is able to penetrate ghosts but not intact RBCs. B) Rapid dilution of the media generates a higher fluorescence contrast inside ghosts, confirming that the dextran is present in the lumen of ghosts. C) Binding of Fl-E5-TAT to ghosts is maintained after washing at pH 7. Ghosts were formed by treating RBCs with 3 µM Fl-E5-TAT at pH 4.5. The ghosts were then washed with PBS at pH 7.0 and imaged by fluorescence microscopy. Staining of the ghosts by the peptide was not reduced by repeating the washes or with time. Similar results were obtained when the pH of the PBS was 4.5 (data not shown).