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. 2019 Aug 19;9:12009. doi: 10.1038/s41598-019-48519-0

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© The Author(s) 2019

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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Exposure to ephrin-A5 increases surface enriched Neogenin protein levels and netrin-1 binding to LMC growth cones. Examples of surface-enriched Neogenin in MN media (a) or ephrin-A5 50 ng/mL (b) treated LMC growth cones. (c) Quantification of surface enriched Neogenin IF in LMC growth cones treated with either MN media or ephrin-A5 50 ng/mL for 20′, ephrin-A5 increases surface enriched Neogenin IF 1.6 ± 0.2-fold, p = 0.037. (d,e) Examples of netrin-1 IF in LMC growth cones treated with either netrin-1 (d) or a combination of netrin-1 and ephrin-A5 (100 ng/mL). (f) Quantification of netrin-1 IF in growth cones treated with netrin-1 or netrin-1 + ephrin-A5, addition of ephrin-A5 results in a 1.5 ± 0.1-fold increase in netrin-1 IF (p = 0.0003). (d) Graph depicting the distribution of netrin-1 IF in growth cones of explants treated with either MN media, ephrin-A5, netrin-1 or netrin-1 + ephrin-A5. Bin #1 coincides with the geometric centre of the growth cone and Bin# 99 coincides with the perimeter of the growth cone. Data are shown as mean ± SEM, statistical significance was tested using a two-tailed unpaired sample t-test. (c) N = 4 (f,g) MN media: N = 3; netrin-1: N = 6; ephrin-A5: N = 6; netrin-1 + ephrin-A5: N = 6.