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. 2019 Jan 24;9:544. doi: 10.1038/s41598-018-35811-8

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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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Co-localization of internalized aSyn species with markers of endosomal/lysosomal pathway. H4 cells were exposed to monomeric (M), oligomeric (O), and fibrillar (F) aSyn. Co-localization of exogenous aSyn (green) with markers of endosomal/lysosomal pathway (red), was analyzed by ICC and confocal microscopy. Analyzed markers included: EEA1 for early endosomes, Rab11 for recycling endosomes, Lamp1/Lamp2a for late endosomes and lysosomal membrane protein; p62 for autophagosomes and lysosomes, and LysoT for lysosomes. A selected region in each image is marked by a dashed frame and its enlarged view is shown at the top left corner of each image. Co-localization of aSyn and endosomal/lysosomal makers (yellow) in selected regions is highlighted by arrowheads and confirmed by the profile intensity plots (Supplementary Fig. S6). Quantification of the degree of co-localization between exogenous aSyn and endosomal/lysosomal markers as well as transferrin (right) demonstrates a generally lower co-localization degree of aggregated aSyn with p62 and LysoT as compared to the co-localization of aSyn with other markers (n = 3, One-way ANOVA with Sidak’s multiple comparisons test).