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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. 5 — a Western blot indicating PARP1 (top, left), SET8 (middle, left), and H4K20me1 (middle, left) levels in total protein extracts from HeLa cells synchronized in G1, S, and G2/M phases, respectively. Western blot of CDT1 protein levels is shown as a cell cycle synchronization control as well as Ponceau stain for loading control and densitometry analyses (bottom, left). Respective densitometry analyses of H4K20me1 (top, right; n = 2) and SET8 (bottom, right; n = 2) relative protein abundances are shown (right) and representative of at least 2 biological experiments. b SET8 immunoprecipitation from total protein extract in HeLa cells synchronized in G1, S, and G2/M phases. Western blots detection of PARP1 (top, left) as well as SET8 immunoprecipitated protein levels (bottom, left) are revealed. Densitometry analyses of PARP1/SET8 ratio during G1, S, and G2/M cell cycle phases are shown (right; n = 3) and are representative of at least 2 biological experiments. c SET8 immunoprecipitation from total protein extract in HeLa cells synchronized in G1, S, and G2/M phases. Western blots detection of ADP-ribosylation as well as SET8 protein levels in SET8 immunoprecipitates (top panel). Respective densitometry analyses of SET8 ADP-ribosylation abundance representative of at least 2 biological experiments are shown (bottom panel; n = 2). d Pulsed chased cells with 5-ethynyl-2′-deoxyuridine (EdU) to label DNA (magenta) is transfected with GFP-SET8 (green). Endogenous ADP-ribose (red) is revealed by anti-ADP-ribose conjugated with Texas Red. Merged images demonstrate the colocalization of EDU, SET8 and ADP-ribose.