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. 2023 Nov 13;26(12):2104–2121. doi: 10.1038/s41593-023-01480-8

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© The Author(s) 2023

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. 3 — a, Diagram of Tau-488 uptake assay. Neurons treated with either Tau-488 alone (left) or Tau-488 together with 100 µg ml⁻¹ heparin (right) before flow cytometry analysis. b, Measurement of individual neuronal Tau-488 uptake (25 nM, 1-h incubation) based on MFI per cell in human neurons. c, Measurement of Tau-488 uptake (25 nM, 1-h incubation) based on percent Tau-488⁺ human neurons. In b,c, n = independent experiments and normalized to E4 MFI (b) or uptake (%) (c). E4, n = 13; E4+heparin, n = 4; E3, n = 4; E3+heparin, n = 4; E4-S/S, n = 4; E4-S/S+heparin, n = 4; E4-R/S, n = 4; E4-R/S+heparin, n = 4; EKO, n = 3; EKO+heparin, n = 4. Analysis was performed on a live cell population of estimated 5,000 cells for each sample. d, Experimental design for long-term heparin treatment of human neurons. e,f, Representative western blot images (e) and quantification of PHF1⁺ p-Tau levels (f) in lysates of E4, E3, E4-S/S or E4-R/S neurons under long-term heparin treatment. In f, PHF1⁺ p-Tau levels were normalized to those of E4. TUJ1 was used as loading control (E4, n = 16; E4+heparin, n = 16; E3, n = 16; E3+heparin, n = 15; E4-S/S, n = 32; E4-S/S+heparin, n = 32; E4-R/S, n = 16; E4-R/S+heparin, n = 16). g, Experimental design for E4 neuron-conditioned medium treatment of human neurons with different APOE genotypes. h,i, Representative western blot images (h) and quantification of PHF1⁺ p-Tau levels (i) in lysates of E4, E3, E4-S/S or E4-R/S neurons after E4 neuron-conditioned medium treatment. In i, PHF1⁺ p-Tau levels were normalized to those of E4. TUJ1 was used as loading control (E4, n = 5; E3, n = 5; E4-S/S, n = 10; E4-R/S, n = 10). In f,i, n =biological replicates. Throughout, data are expressed as mean ± s.e.m. Differences between groups were determined by two-way ANOVA followed by Tukey’s multiple comparison test (b,c,f) or ordinary one-way ANOVA followed by Tukey’s multiple comparison test (i). Some comparisons of P ≤ 0.05 are labeled on the graph. Hep, heparin; fluor, fluorescence.

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