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. 2020 Jul 7;11:3391. doi: 10.1038/s41467-020-17069-9

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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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Fig. 3 — a Endogenous levels of ADP-ribosylation were analysed by indirect immunofluorescence in wild-type and ARH3^−/− U2OS cells by detergent pre-extraction prior to fixation and staining with anti-mono-ADP-ribose binding reagent, anti-poly-ADP-ribose antibody, anti-poly-ADP-ribose binding reagent (br), and anti-PAN-ADP-ribose binding reagent. Representative ScanR images (left) and quantification (right) are shown. b Endogenous chromatin ADP-ribosylation in control and patient fibroblasts measured after detergent pre-extraction and staining with anti-PAN-ADP-ribose binding reagent or anti-poly-ADP-ribose antibody. Quantification (right) and representative images (left) are shown; scale bar, 10 μM. Quantification for a and b was done by using ScanR software. Data are the mean ± SEM of n = 5 for a and n = 3 for (b) biologically independent experiments. Statistical analysis (two-tailed t-test) is indicated (*P < 0.05; **P < 0.01; ns not significant). c Endogenous ADP-ribosylation in control, ARH3 patient and XRCC1 patient fibroblasts was detected by western blotting using anti-PAN-ADP-ribose binding reagent or anti-poly-ADP-ribose antibody. The arrow denotes mono(ADP-ribosylated) histones and the black asterisk is a nonspecific band. d Endogenous ADP-ribosylation in wild-type, PARP1^−/− and ARH3^−/− U2OS cells was detected by western blotting using anti-PAN-ADP-ribose binding reagent, anti-poly-ADP-ribose antibody, or anti-mono-ADP-ribose binding reagent. Arrows and the black asterisk are as above, and the red asterisk denotes poly(ADP-ribosylated) proteins (most likely ribosylated PARP1) not bound to chromatin.