Anti snap 25

Anti-SNAP-25, anti-actin, anti-Flag, anti-GST, anti-syntaxin, peroxidase conjugated anti-rabbit IgG and anti-chicken IgG were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-SNAP-25^pThr138 (Abgent, San Diego, CA, USA), anti-MYPT1^1-296 [20 (link)], anti-PP1cδ (Millipore, Billerica, MA, USA), anti-GAPDH (Santa Cruz, CA, USA), anti-CPI17^pThr38 [22 (link)], horseradish peroxidase-linked anti-mouse IgG (Cell Signalling, Danvers, MA, USA), Clean-Blot IP Detection Reagent (Thermo Scientific, Waltham, MA, USA) and Texas Red-X phalloidin (Life Technologies, Carlsbad, CA, USA) were purchased as indicated. Alexa Fluor 488-conjugated anti-rabbit IgG, Alexa Fluor 546-conjugated anti-mouse IgG, Alexa Fluor 546-conjugated anti-goat IgG and To-Pro-3 iodide were obtained from Molecular Probes (Eugene, OR, USA).

The activity of rBoNT/E-LC was determined by in vitro endopeptidase assay using His-tag rSNAP-25 substrate. The cleavage activity was performed in dose dependent manner in a final reaction volume of 20 µL. This assay was performed by incubating the fixed concentration rSNAP-25 of 5.0 nM with varying concentration of rBoNT/E-LC in reaction buffer (25 mM Tris, 100 mM NaCl, 19.2 mM glycine, 100 μg/mL BSA, 0.1 mM dithiothreitol (DTT), 10 μM ZnCl2, pH 7.5) and incubated for 30 min at 37 °C and the reaction was stopped by adding the 4 × dye sample lysis buffer and boiled at 100 °C for 5 min. The reactions were resolved by 13% SDS-PAGE followed by western blot using monoclonal antibody rabbit anti-SNAP-25 (Sigma Aldrich, USA) in a dilution of 1:5,000, as primary antibody. The goat anti rabbit HRP conjugate antibody was used as secondary antibody in a dilution of 1:20,000. The reactions were detected by chemiluminescence using an ECL western blot kit (Biological industry, Israel) as per manufacturer's instructions. Film was exposed for 15 s before development. The percentage of cleaved substrate was measured by densitometry. The recombinant SNAP-25 vector was transformed into BL21DE3 host cells and rSNAP-25 was purified as abovementioned method for light chain under native conditions and used as substrate.