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Recent biochemical and molecular genetic studies have established synaptic vesicle glycoprotein 2C (SV2c) as the protein receptor for Botulinum Neurotoxin Type A (BoNT/A). The luminal domain loop of SV2c, between transmembrane domains 7 and 8, has been shown to be the location of BoNT/A binding. Utilizing this information, we have shown through GST pull-down experiments that we can detect binding...
The DARET (Depolarization After Resonance Energy Transfer) assay is a homogenous, coupled FRET-fluorescence polarization (FRET-FP) assay for BoNT/A or /E proteolytic activity that relies on a fully recombinant substrate. The substrate consists of BFP and GFP proteins flanking SNAP-25 residues 134–206. In the assay, the substrate is excited with plane-polarized light at 379nm, which primarily excites...
Pasteurella multocida toxin (PMT) stimulates and subsequently uncouples phospholipase C β1 (PLCβ1) signal transduction through its selective action on the α subunit of the Gq-protein. Here, we describe the application of an NFAT-β-lactamase reporter assay as a functional readout for PMT-induced activation of the Gq-protein-coupled PLCβ1-IP 3 -Ca 2+ signaling pathway. Use of the NFAT-β-lactamase...
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