Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2017 Nov 21;8:1637. doi: 10.1038/s41467-017-01534-z

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2017

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 1 — Lacking both a-series and b-series of complex gangliosides reduced binding and entry of BoNT/C1 and D into neurons, but did not affect BoNT/DC. a A schematic drawing of ganglioside synthesis pathways, with the step blocked in Sial9 KO mice marked. b WT and Sial9 KO cortical neurons were exposed to BoNT/C1 (100 nM, 5 min in High-K⁺ buffer). Cells were washed, fixed, and subjected to immunostaining analysis using a polyclonal anti-BoNT/C1 antibody (green). SV2 was co-stained in parallel using a pan-SV2 monoclonal antibody (red) to mark presynaptic terminals. Binding of BoNT/C1 is largely abolished in Sial9 KO neurons. Scale bar here and thereafter represents 20 µm. c Experiments were carried out as described in b, except that neurons were exposed to HA-tagged BoNT/D-H_C (100 nM). Binding of BoNT/D-H_C was detected with a monoclonal HA antibody (green). Synapsin was co-stained to mark presynaptic terminals (red). Binding of BoNT/D-H_C is largely abolished in Sial9 KO neurons. d Experiments were carried out as described in c, except that neurons were exposed to HA-tagged BoNT/DC-H_C (100 nM). BoNT/DC-H_C binds equally well to WT and Sial9 KO neurons. e WT and Sial9 KO neurons were exposed to the indicated concentrations of BoNT/C1 (5 min in High-K⁺ buffer). Neurons were washed and further incubated in toxin-free medium for 8 h. Neurons were harvested and cell lysates were subjected to immunoblot analysis. Cleavage of SNAP-25 by BoNT/C1 generates a slightly smaller fragment, marked with an asterisk. Entry of BoNT/C1 into Sial9 KO neurons was reduced, as there is less cleavage of SNAP-25 and syntaxin 1. f, g Experiments were carried out as described in e, except that neurons were exposed to BoNT/D (f) or BoNT/DC (g). Cleavage of VAMP2 by BoNT/D or BoNT/DC results in disappearance of its immunoblot signals. Entry of BoNT/D into Sial9 KO neurons was blocked, as there was no cleavage of VAMP2 in Sial9 KO neurons. Entry of BoNT/DC into WT and Sial9 KO neurons was at similar levels. One of two (b–d) or three (e–g) independent experiments is shown