BoNT/A LC Inhibition Assay

2 μM of BoNT/A LC (2X final concentration) was pre-incubated with 2X the indicated final concentration of each compound at room temperature for 30 min in 20 μL of Tris buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl). To each BoNT/A LC and selenide compound mixture, 20 μL of approximately 2 mg ml⁻¹ rat brain detergent extract (prepared as in Zhang, et al^{23 (link)}) was then added and incubated at 37 °C for 1 hour. Samples were resolved via SDS-PAGE, with full-length and cleaved SNAP-25 substrate measured via western blot with mouse monoclonal antibodies for SNAP-25 (71.1, 1:2,000 dilution, Synaptic Systems) and actin (AC-15, 1:1,000, Sigma) to measure loading.