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. 2017 Oct 27;8:1164. doi: 10.1038/s41467-017-01283-z

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© 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/.

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Fig. 1 — EphB1 is upregulated in axotomised neurons, where astrocytes display ephrin-B1 IR. a Schematic diagram demonstrating right-sided unilateral facial nerve (Fn) axotomy (Ax) and the associated ipsilateral (IL) facial motor nucleus (FMN) in which immunofluorescence was analysed in comparison with the non-axotomised contralateral (CL) FMN. b Representative immunofluorescence images showing abundant EphB1 IR 14 days after axotomy in the IL FMN in which 98% of neurons are defined as motor neurons³⁴. c Images showing EphB1 and NeuN labelling and also ephrin-B1 and GFAP IR in corresponding neurons or astrocytes (ACs), respectively. d High magnification images demonstrate cell surface labelling for EphB1 in sections immunostained before permeabilisation and also cytoplasmic EphB1 IR post-permeabilisation in a ChAT-positive motor neuron (MN) (see also Supplementary Fig. 1) and NeuN positive neurons with motor neuron morphology³² in the middle panel. In the lower panel ephrin-B1 positivity is demonstrated in GFAP-labelled astrocyte soma and processes (see also Supplementary Fig. 1). e Graph showing the mean of fold changes in the proportion of EphB1-positive neurons in the IL FMN normalised to the CL FMN following axotomy (1, 7, 14, 28 days; n = 3 per time point, respectively, and 3–4 brainstem sections in each group; *p = 0.016, *p = 0.046 compared to values at day 1, F = 9.431; one-way ANOVA with Dunnett’s post hoc test; see also Supplementary Fig. 1). f Western blot (WB) showing EphB1, ephrin-B1 and pSTAT3 protein levels (both 79 and 86 kDa isoforms) in tissue lysates from both the IL and CL FMN of three WT mice at day 14 post-axotomy (see also Supplementary Fig. 8). g Bar graph represent relative WB band densities of EphB1, ephrin-B1 and pSTAT3 in the IL FMN and is expressed as fold increase over the band density levels for the CL FMN after normalisation to β-actin (n = 3 mice, unpaired t-test, **p = 0.006, ***p = 0.0004, *p = 0.034). Data presented as mean ± SEM. Scale bars: 50 μm for (b), 20 μm for (c) and 10 μm for (d)