Western Blot Analysis of BoNT-Intoxicated Neurons

For the western blot analysis shown in Fig 1, fully differentiated mouse ES-MNs were treated with the compounds (20 μM) (Fig 2) 30 min prior to the addition of 250pM BoNT/A. For hES-MN pre-intoxication models shown in Figs 3A, 4A and 5A, fully differentiated human ES-MNs (Day 35) were treated with the titrated concentrations of compounds for 30 min and then intoxicated with either BoNT/A, BoNT/B or trypsin-activated BoNT/E (MetaBiologics, Madison, WI), and incubated at 37^°C for 4 hr. Bafilomycin was utilized as a positive control [30 (link)]. For post-intoxication studies, cells were intoxicated with either BoNT/A, BoNT/B or trypsin-activated BoNT/E for either 30 or 60 min and then 20 μM compound was added to the cultures. Total intoxication time was kept constant at 4 hr for all immunoblotting analyses. Following intoxication, samples were prepared and BoNT mediated SNARE protein cleavage was quantified using standard immunoblotting procedures employing SNAP-25 (Covance) and VAMP-2 (R&D Systems) antibodies—as described previously [30 (link), 36 (link), 37 (link)]. The reported values were calculated from at least three independent experiments.