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. 2020 May 14;10:7959. doi: 10.1038/s41598-020-64868-7

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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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Primary screen for activators of FANCD2 mono-ubiquitination in the absence of FANC-C -E -F. (a) All compounds screened in duplicate plotted as Z scores and presented on a per library representation. (b) Nine-point titration of 47 activator hits. (c) Area under curve plot calculated from the curves in 3b, demonstrating activation potency for each compound. (d) Cropped images from western blot analysis of 4 activator hits for an effect on the FANCD2 mono-ubiquitination assay with recombinant protein. Positive (Complete Core Complex) and negative (DMSO) controls are shown on the far-left panel. The four activator hits from the secondary screen were analysed with FANCD2-ubiquitination assay (without FANC-E-F) visualised on western blot by probing with streptavidin to detect biotinylated-ubiquitin. Three concentrations of the compounds were used; 1 µM, 10 µM and 100 µM.