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. 2018 Sep 28;9:3986. doi: 10.1038/s41467-018-06507-4

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© The Author(s) 2018

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/.

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Fig. 5 — Dominant-negative activity of mutant Munc18-1 on wild-type Munc18-1. a Protein levels of Munc18-1 in primary neurons. Levels of GFP-tagged WT or mutant Munc18-1 and endogenous Munc18-1 in heterozygous Munc18-1 neurons were analyzed by quantitative immunoblotting, normalized to α-tubulin levels. Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test; n = 3 independent experiments). b Same as in a, except that myc-tagged Munc18-1 variants were co-expressed with HA-tagged WT Munc18-1 in Munc18-1 knockout neurons. Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test; n = 5–6 independent experiments). c Co-immunoprecipitation of mutant with wild-type Munc18-1. Lysates of HEK293T cells that were co-transfected with HA-tagged WT and myc-tagged mutant Munc18-1 were subjected to immunoprecipitation with an anti-c-myc antibody (IP) or no antibody (control). Precipitated myc- and HA-tagged Munc18-1 was analyzed with the respective input by immunoblotting. GAPDH served as control. d Solubility of Munc18-1. Primary neurons infected as in b were solubilized in 0.1% Triton X-100 (TX). Equal volumes of TX-soluble and -insoluble fractions were analyzed by quantitative immunoblotting. α-Tubulin served as control. Data are means ± SEM (*p < 0.05, ***p < 0.001 by Student’s t test; n = 5–7 independent experiments). e Locomotion of C. elegans. Body bends per 30 s were counted. Data are means ± SEM (*p < 0.05, **p < 0.01 by Student’s t test; n = 15 independent experiments on ten worms per experiment). f Heat-induced paralysis. Paralysis at 37 °C was scored at indicated time points. Data are means ± SEM (***p < 0.001 by two-way ANOVA, compared to N2; n = 3 independent experiments on ten worms per experiment). g Mutants display reduced acetylcholine release at the worm neuromuscular junction. Paralysis of N2 worms expressing WT or mutant unc-18 was measured after 60 min exposure to aldicarb. Data are means ± SEM (**p < 0.01, ***p < 0.001 by Student’s t test; n = 6 independent experiments on 20 worms per experiment)