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. 2019 Jul 23;9:10669. doi: 10.1038/s41598-019-46956-5

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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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MTP18 is a mitochondria associated protein that regulates mitochondrial size in RGCs. (a) Western blot of RGC lysates from 3 replicate RGC purifications probed for MTP18 and GAPDH as a loading control. RGCs were purified from P4 rat retinas. Blots were cut, individually probed with antibodies and then imaged. (b) Western blot of a mitochondrial enriched fraction (mito.) cytosolic fraction (cytosolic), and crude total cell lysate fraction (lysate) from adult rat retinas. Samples probed using an MTP18 antibody and Cytochrome c oxidase subunit 1 antibody (MTCO1), to validate mitochondrial enrichment. Images were captured from the same blots, sequentially probed with antibodies. (c) MTP18 mRNA expression after AAV2 mediated expression of Anti-luciferase (Anti-Luc), Anti-MTP18, and MTP18 expression vectors in P4 RGCs. Data captured by QRT-PCR, normalized to GAPDH and expressed as mean expression relative to Anti-luciferase (N = 3 replicate treatments, significance ascribed by one-way ANOVA and Tukey multiple comparison, *p ≤ 0.05). (d) TEM images of mitochondria in P4 RGCs neurites treated with indicated AAV2 viruses or combinations of virus (scale bar 1 µm). (e) Mean area of mitochondria identified in P4 RGC neuritis are graphed for each representative AAV2 treatment group, N ≥ 15 de-identified neurite images selected from 3 replicate viral treatments per group (significance determined by one-way ANOVA and Tukey multiple comparison, *p ≤ 0.05). All error bars represent + SEM. Full length blots are available in Supplemental Fig. 3.