hES-MN cultures (day 35) were intoxicated with increasing concentrations of either BoNT/A or BoNT/B or trypsin-activated BoNT/E (MetaBiologics) and incubated at 37 °C for 4 h (Fig. 3). Following intoxication, samples were processed, and SNAP-25 (Covance) and VAMP-2 (R&D Systems) protein cleavages were quantified using standard immunoblotting procedures—as described previously (Huang et al. 2011 (link); Pellett et al. 2007 (link)). Quantification of changes in total VAMP-2 protein levels was calculated by normalizing the total VAMP-2 band intensity values to corresponding GAPDH levels relative to non-toxin treated control conditions run on each gel. For inhibitor studies, Triticium vulgaris Lectin (TVL) and bafilomycin were used at titrated concentrations and added to the cultures 30 min prior to intoxication. Both reagents were obtained from Sigma. An antibody that neutralizes BoNT/A (4A2-4) (produced at the US Army Medical Research Institute of Infectious Diseases) and the control antibody (Anti staphylococcal enterotoxin B) (Toxin Technology) were simultaneously applied with 1 nM BoNT/A to the cultures.