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. 2022 Nov 25;5:1292. doi: 10.1038/s42003-022-04241-8

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© The Author(s) 2022

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 — a Co-immunoprecipitation of endogenous SET8 and PARP1 in HCT116 cells. Rabbit IgG (left lane) was used as a negative control. Non-specific bands are represented as NS. b Colocalization between FLAG-PARP1 (red) and GFP-SET8 (green) during cell cycle in COS-7 cells. DAPI (blue) represents the nuclear DNA content. c, d Mapping of domain interactions using GST-pulldown assays between SET8 domains (top, left) and full-length recombinant PARP1 protein and GST-pulldown assays between PARP1 domains and full-length recombinant SET8 protein (top, right). The PARP1 or SET8 binding were detected by western blotting using PARP1 antibody (middle, left) or SET8 antibody (middle, right), respectively. Ponceau stain gels (bottom) represent the amount of GST beads constructs used for the GST-pulldown assays. e In vitro detection of full-length recombinant SET8 ADP-ribosylation by full-length recombinant PARP1 by western blotting using anti-ADP ribose antibody (top). Ponceau stain gels (bottom) represent the amount of PARP1 and SET8 recombinant enzyme used for ADP-ribosylation assay (bottom). f Detection of SET8 lysines ADP-ribosylation using mass spectrometry analysis of SET8 ADP-ribosylated peptides by full-length recombinant PARP1 protein in vitro.