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. 2017 Nov 28;7:16477. doi: 10.1038/s41598-017-16613-w

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© The Author(s) 2017

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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Ecto-ART expression and activity on microglia from mixed glial cell cultures. (a) Reactivity of fluorochrome-conjugated ARTC2.1 and ARTC2.2-specific mAbs was analyzed by flow cytometry of BALB/c WT spleen CD3⁺ T cells, F4/80⁺ macrophages (MΦ). or CD11b⁺GLAST⁻ microglia from BALB/c WT mixed glial cell cultures. BALB/c ARTC2.1^−/− and ARTC2.2^−/− cells were used as negative controls. (b) Etheno-NAD (eNAD⁺) can be used as surrogate substrate for ARTC2, which etheno-ADP-ribosylates cell surface proteins. This can be detected by use of etheno-adenosine-specific mAb 1G4. Ecto-ART activity was analyzed by measuring etheno-ADP-ribosylation of cell surface proteins on BALB/c WT and ARTC2.1^−/− spleen MΦ (upper panel) and on microglia from mixed glial cell cultures (lower panel) after incubating cells with 100 µM eNAD⁺ (blue), with 100 µM eNAD⁺ and 2 mM DTT (red) or without additions (grey). Etheno-ADP-ribosylated proteins were detected with mAb 1G4. Data are representative of 3 independent experiments.