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. 2020 Jun 16;10:9752. doi: 10.1038/s41598-020-66522-8

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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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Segregation between LecA and StxB can be reconstituted in GUVs containing a natural mixture of Gb3 species. GUVs containing 5 mol-% Gb3-mix were incubated with LecA-Al647 (200 nM, orange) and StxB-Cy3 (600 nM in A and 200 nM in B, blue) (A,B). The images show equatorial sections through representative GUVs with a diagram displaying the corresponding circular intensity profiles underneath. Phase-separated GUV (A) contained DOPC/cholesterol/sphingomyelin/Gb3-mix in the ratio 42.5/14.5/42.5/5 mol-%, whereas non-phase-separated GUV (B) contained DOPC/cholesterol/Gb3-mix in the ratio 64.5/30/5 mol-%. GUVs in (A,B) were additionally spiked with 0.5 mol-% of the membrane marker β-BODIPY-FL-C₅-HPC (green). Scale bars correspond to 5 μm.