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. 2018 Sep 26;9:3936. doi: 10.1038/s41467-018-06197-y

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© The Author(s) 2018

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 — UBL3 modification influences the sorting of proteins to the sEV. a, b Heat map of z-scored LFQ intensities of the significantly regulated proteins (ANOVA, FDR = 0.05 & S0 = 1) in all three conditions (3xFlag-UBL3, 3xFlag-UBL3C113/114A or 3xFlag empty vector) revealed the UBL3 interacting proteins (Cluster 1). The colour key denotes normalised protein abundances (z-score). Profiles of all proteins (1447) found in Cluster 1 are shown and 454 of those (31%) with ‘extracellular vesicular exosome’ annotations are highlighted in dark blue. c Volcano plots showing the p-values vs. the log2 protein abundance differences in Flag-UBL3 compared with either Flag-UBL3C113/114A or Flag empty vector. The significance cut-off is based on an FDR = 0.05 and S0 = 1. Disease-related molecules, black colour. HRAS and KRAS, red colour. TUBA1A, 1B, 1C, and 4A, orange colour. TUBB, B2A, B3, B4A, B4B, B6, and B8, blue colour. d sEV pellets were blotted with anti-Ras antibodies. e Images of phosphorylated ERK (pERK) in PKH67-labelled sEV-incorporated MDA-MB-231 cells. Purified sEVs from the conditioned medium of MDA-MB-231 cells transfected with RasG12V and either mock, 3xFlag-UBL3, or 3xFlag-UBL3C113/114A or with mock and 3xFlag-UBL3 were labelled with PKH67 dye (green) and added to MDA-MB-231 cells. Scale bars, 10 μm. f Each plot shows pERK fluorescence in PKH67-labelled sEV-incorporated cells normalised to the average pERK values in the two neighbouring PKH67-labelled sEV-unincorporated cells from each image in e. RasG12V-mock, n = 18; RasG12V-UBL3, n = 19; RasG12V-UBL3C113/114A, n = 19; Mock-UBL3, n = 21. **, p < 0.01; ***, p < 0.001 by Kruskal–Wallis/Dunn's multiple-comparisons test