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. 2021 May 10;12(5):466. doi: 10.1038/s41419-021-03710-y

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© The Author(s) 2021

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. 4 — a Representative DAB immunohistochemistry (Left panel) and immunofluorescence staining (right panel) using HA antibody in lumbar spinal cord α-MNs of wt and P56S-VAPB tg mice, showing globular aggregates in the α-MNs (arrowheads) of P56S-VAPB tg mice. Scale bars: 15 µm. b, c Double immunofluorescence labelling for HA and for the autophagy marker LC3 (b) and p62 lower panel showing co-localisation (arrowheads) with P56S-VAPB aggregates (c), and DAB immunohistochemistry showing globular accumulations of p62 (arrows, upper panel) (c) in lumbar spinal cord α-MNs, Scale bars: 15 µm. d Immunoblot analysis of lumbar spinal cord homogenates from P56S-VAPB (n = 6, male, 200 days) and their age-matched wt-VAPB mice (n = 3, male, 201 days), showing an overall increased level of ER stress markers pIF2α and autophagy markers p62 and LC3 in P56S-VAPB tg mice. Corresponding densitometric data are shown at the bottom; representing the average relative band intensity from n = 3 wt-VAPB and n = 6 P56S-VAPB of p62, LC3-II/LC3-I normalised with tubulin levels. Unpaired Student’s t test for comparison between these two sample groups (average, n = 3 wt-VAPB mice and average, n = 6 P56S-VAPB mice) Values were expressed as mean ± standard error of mean (SEM) from three independent blots. The asterisks (*) denote significant differences (*p < 0.05). e–g Double immunofluorescence labelling for HA and for the SNARE protein STX17 (e), early autophagy proteins ULK1 (f) and ATG5-ATG12 (g) showing their co-localisation (arrowheads) with P56S-VAPB aggregates in lumbar spinal cord α-MNs. Note the loss of STX17 immunoreactivity (arrows in e) in α-MNs harbouring P56S-VAPB aggregates in lumbar spinal cord of P56S-VAPB mice. Scale bars: 15 µm.