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. 2020 May 4;11(5):314. doi: 10.1038/s41419-020-2503-3

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© The Author(s) 2020

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, b Primary neurons (DIV2) were applied Accell^TM SMARTpool siRNA for non-targeting (NT) or Ext2 gene (NM_001355075) at concentrations of 50–500 nM for 96 h. 18s rRNA was used for the reference gene and normalization in gene expression analysis. Ext2 siRNA (500 nM) significantly reduced the exostosin-2 mRNA (a) and protein (b) levels compared with NT siRNA. Results were from three independent experiments and shown as fold difference expression relative to control (mean ± SEM). Statistical analyses were measured by one-way ANOVA with Tukey’s test (a) or unpaired, two-tailed Student’s t test (b) from three biological independent experiment. Results showed as the value of mean ± SEM, a ****p < 0.0001 vs UT group, ^##p < 0.01, ^####p < 0.0001 vs NT group, b **p < 0.01 vs NT group. c–e Neurons (DIV2) were preincubated with NT or Ext2 siRNA for 96 h followed by AF568-tagged TauO from AD (c), PSP (d), or DLB (e) treatment for 18 h. Cells were immunolabeled with a mature neuronal marker (βIII-tubulin, blue), and an early endosomal marker (Rab5, green). Representative orthogonal images depicted AF568-tagged TauO co-localized to early endosomes (arrows). Scale bar: 2 and 10 μm. f–h Neurons were untreated (UT) or treated with 0.1 μM biotin-tagged TauO (+) from AD (f), PSP (g), or DLB (h) with similar experimental conditions and parameters as for (c–e). Internalized tau was detected using anti-Streptavidin antibody. Representative Western blot images depicted the appearance of exogenously applied TauO. Internal controls from the same blot were probed with anti-βIII-tubulin. Analysis of internalized tau levels was on the lower panel of each immunoblot showing as Streptavidin band intensity (HMW: 75–250 kDa) normalized to internal control and presented as the percentage of UT group. Statistical analyses were measured by one-way ANOVA with Tukey’s test from three biological independent experiments. Results showed as the value of mean ± SEM, **p < 0.01, ***p < 0.001 vs UT group. ^##p < 0.01 vs TauO-treated group. Western blot analyses of TauO from AD, PSP, or DLB were performed on separate membranes. The same membranes were re-probed for marker proteins of autophagy–lysosomal pathway as shown in Fig. 5a–c. The immunoblots for internal controls shown in Fig. 4f–h were reused in Fig. 5a–c.