Fig. 3. Cleavage of VAMP-2 by the clostridial neurotoxins TeNT: in vitro cleavage. Membrane fractions enriched in intracellular vesicles from CD8 cells and rabbit brain membranes were incubated with TeNT 500 nM for 1 hour at 37°C. TeNT was previously activated by incubation with 10 mM dithiothreitol (DTT) for 2 hours at 37°C. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL-plus). Cleavage of VAMP-2 in intact CD8 cells. CD8 cells were grown to confluency in 10 mm Petri dishes. Cells were incubated in the presence or in the absence of whole TeNT (100 nM, for 3 hours at 37°C in the medium). Proteins were transferred and subjected to western blotting using monoclonal antibodies (1:100 dilution) against human VAMP-2. The results shown are representative of at least three separate experiments. Internalization of TeNT-FITC was visualized by immunofluorescence analysis. Intact cells were exposed to TeNT-FITC, fixed and examined at the fluorescence microscope. TeNT-FITC was internalized in intracellular structures. Bar, 8 µm.