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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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Investigations how Gb3 species and phase separation influence LecA and StxB binding efficiencies. GUVs doped with 0.5 mol-% of the membrane marker β-BODIPY-FL-C₅-HPC (green) and 5 mol-% Gb3-mix (A,B) or 5 mol-% Gb3-C24:0 (C,D) were incubated with LecA-Cy5 (200 nM, orange) or StxB-Cy5 (200 nM, blue). Non-phase-separated GUVs ((A,C) contained DOPC/cholesterol/Gb3 in the ratio 64.5/30/5 mol-%, whereas phase-separated GUVs (B,D) consisted of DOPC/cholesterol/sphingomyelin/Gb3 in the ratio 42.5/14.5/42.5/5 mol-%. The images A – D show equatorial sections through representative GUVs. Scale bars correspond to 5 μm. (E,F) Quantitative analysis of the binding efficiencies of LecA (E) and StxB (F) to non-phase-separated GUVs (red) or the ld (green) or lo (blue) phase of phase-separated GUVs containing 5 mol-% of either Gb3-mix (mix), Gb3-C24:0 (C24:0), Gb3-C24:1 (C24:1), or Gb3-FSL (FSL). For each condition, the middle horizontal line represents the median, the boxes the 25^th to 75^th percentiles, and the whiskers the min and max values. In Table S1 the descriptive statistics of the data are summarized and Table S2 contains the data from a significance analysis.