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. 2016 Nov 1;27(21):3329–3341. doi: 10.1091/mbc.E16-03-0184

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© 2016 Toft-Bertelsen et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 6: — Increasing syntaxin-1 immunoavailability reduces calcium currents in mouse chromaffin cell. (A) Confocal midsections of WT and Doc2B KO adrenal chromaffin cells with and without Doc2B overexpression (o.e.) stained against syntaxin-1. Scale bar, 5 μm. (B) Quantification of the syntaxin-1 immunolabeling indicates a very strong increase upon Doc2B overexpression (note broken axis). ***p < 0.001 (one-way ANOVA with Tukey’s post hoc test). N = 2, n = 21–26. (C) Amplitudes of calcium currents in Doc2B KO cells with and without Doc2B overexpression. (D) Western blotting of cultured chromaffin cells shows that the endogenous expression level of syntaxin-1 is not changed upon overexpression of Doc2B. (E) Confocal midsections of SNAP-25 WT cells with and without ubMunc13-2 overexpression stained for syntaxin-1. Scale bar, 5 μm. (F) Quantification of the immunoavailability of syntaxin-1 reveals an increase upon ubMunc13-2 overexpression. *p < 0.05 (Student’s t test, two-tailed). N = 2, n = 20. (G) Calcium currents in SNAP-25 WT cells and SNAP-25 WT overexpressing ubMunc13-2. ***p < 0.001 (Student’s t test, two-tailed). N = 2, n = 20. (H) Confocal midsections of Munc18-1 KO cells with and without Munc18-1 overexpression, stained for syntaxin-1. Scale bar, 5 μm. (I) Quantification of total syntaxin-1 fluorescence intensity. (J) Calcium currents in Munc18-1 KO cells with and without Munc18-1 overexpression. Means ± SEM. No significance is found (one-way ANOVA, Tukey’s post hoc test). N = 3, n = 21–23. (K) Western blotting showed that the endogenous expression level of syntaxin-1 is not changed upon overexpression of Munc18-1. (L) Confocal midsections of SNAP-25 WT cells with and without MBCD stained against syntaxin-1. Scale bar, 5 μm. (M) Quantification of syntaxin-1 immunolabeling indicates a strong increase upon treatment with MBCD. ***p < 0.001 (Student’s t test, two-tailed). N = 2, n = 26–28. (N) Amplitudes of calcium currents in SNAP-25 WT cells treated with MBCD were decreased compared with untreated WT cells. ***p < 0.001 (Student’s t test, two-tailed). N = 2, n = 26–28.