TeNT associates with SV2.

<p>(A) Biotinylated BoNT/B, E and TeNT were pre-incubated with neutravidin beads and then mixed with brain detergent extract. Beads were washed and bound material was subjected to SDS-PAGE and immunoblot analysis. BoNT/B and BoNT/E both associated with their receptors, synaptotagmin 1 (syt1, Cl 69.1) and SV2 (pan-SV2), respectively. TeNT showed a strong association with SV2, but not with other synaptic vesicle proteins such as syt1 and synaptoporin (porin). (B) BoNT/E (20 nM) was analyzed for binding to hippocampal neurons in the presence and absence of 2 µM HCR/T. Binding of BoNT/E to neurons was substantially reduced by HCR/T, indicating competition for binding to SV2. (C) Hippocampal neurons were treated with 200 pM BoNT/E in the presence and absence of 20 nM HCR/T. The arrow indicates the BoNT/E cleaved form of SNAP-25. Immunoblot analysis revealed that the presence of HCR/T resulted in the reduced cleavage of SNAP-25 by BoNT/E, again indicating competition for binding to the same receptor.