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. 2020 Nov 17;18(1):556–568. doi: 10.1007/s13311-020-00967-7

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Functional evaluation of BiTox/AA in neuronal cultures. (a, Upper panel) example immunoblot of human SiMa neuroblastoma cells treated with BiTox/AA or BoNT/A for 72 h using an anti-SNAP-25 antibody (n = 3). Note the downward molecular shift of SNAP-25 upon the action of botulinum protease correlating with its increasing doses. Positions of the intact and cleaved SNAP-25 are indicated. (a, Lower panel) example immunoblot of SiMa neuroblastoma cells treated with either 1 nM BiTox/AA or 1 nM BoNT/A for the indicated duration of time using an anti-SNAP-25 antibody (n = 3). (b) Graph showing the change in CMAP amplitude measured by EMG in rats 72 h after injection with varying doses of BiTox/AA and BoNT/A, compared to the baseline amplitude (left panel). Bar chart showing the dose difference in logarithmic scale required to achieve 50% reduction in CMAP values measured 24 h and 72 h post injection (right panel). (c) Example immunoblot showing the specificity of the cSNAP-25 antibody to the cleaved end of SNAP-25, as compared to antibody raised against the whole SNAP-25 protein. (d) Examples of fluorescent micrographs of cultured rat trigeminal neurons treated with either vehicle or BiTox/AA and co-immunostained using the cSNAP-25 antibody and an anti-tubulin antibody. (e) BiTox/AA-cleaved SNAP-25 co-localizes with a subset of CGRP neurons in rat trigeminal neuron culture