Snap 25

Protein from neuronal cultures was harvested, quantified by BCA (Pierce), separated by SDS-PAGE and transferred to PVDF. Blots were stained and imaged as previously described^{19 (link)}. Primary antibodies used for Western blotting included SNAP-25 (Covance, Princeton, NJ), STX1, and SYB2 (Synaptic Systems, Gottingen, Germany). Blots were fluorescently probed using a goat anti-mouse secondary conjugated to Alexa Fluor 488 (Invitrogen, Carlsbad, CA) for detection of STX1 (33 kDa), SYB2 (12 kDa) and SNAP-25 (18, 24 or 25 kDa).

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.