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. 2018 Jul 9;9:2658. doi: 10.1038/s41467-018-05077-9

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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. 1 — Exosomes shuttle mitochondrial proteins. a Differential ultracentrifugation protocol for isolating and purifying exosomes from cell culture supernatants. b Size distribution analysis by Nanoparticle Tracking Analysis (NTA) of purified EVs from primary human T lymphoblasts. c Gene ontology (GO) cellular component analysis of peptides identified in T cell EVs. d Western blot analysis of mitochondrial proteins in EVs obtained from the culture supernatant of primary human T lymphoblasts with or without treatment with phorbol myristate acetate (PMA) plus ionomycin. Cells and EVs were blotted for proteins associated with mtDNA (TFAM and ATAD3), for proteins located in the inner mitochondrial membrane (COX1 and Cytochrome C), the outer mitochondrial membrane (VDAC1), the mitochondrial matrix (mitochondrial manganese superoxide dismutase, MnSOD), and for the exosome markers TSG101 and CD81. e Western blot analysis of ATAD3, TFAM, and the exosome markers CD81 and TSG101 in sucrose fractions. EVs obtained from the culture supernatant of human T lymphoblasts were laid on a discontinuous sucrose gradient and floated by overnight centrifugation. Gradient fractions were collected and analyzed by immunoblot to reveal the distribution of mtDNA-binding proteins and exosomal proteins in the sucrose fractions from lower to higher sucrose density (left to right). Gels shown are representative out of three independent experiments