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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. 3 — a GFP-SET8 immunoprecipitation in overexpressed GFP-SET8 COS-7 cells with or without FLAG-PARP1 overexpression in presence or not of the proteasome inhibitor MG132. Western blots detecting the amount of GFP-SET8 protein overexpressed in total extract (top, left) as well as the amount of FLAG-PARP1 protein overexpressed (top, right) using anti-GFP and anti-FLAG antibody, respectively. Western blots detecting the amount of ubiquitin (Ub) (bottom, left) or ADP-ribosylation (bottom, right) of immunoprecipitated GFP-SET8 protein using anti-HA and anti-ADP ribose antibody, respectively. Anti-actin was used as a loading control (middle). The fold increase was calculated by densitometry and indicated at the bottom of the western blot. b GFP-SET8 immunoprecipitation in overexpressed GFP-SET8 COS-7 cells in presence or not (DMSO) of Cullin inhibitor (Cul4Ai) or PARG inhibitor (PARGi). Western blot detection of total GFP-SET8 protein levels overexpressed in COS-7 cells (1st panel). Anti-actin antibody was used as control (2nd panel). Western blot detecting the amount of poly-ADP ribosylation, using anti-PAR antibody (3rd panel) or the amounts of HA-ubiquitin using anti-HA antibody (4th panel) of immunoprecipitated GFP-SET8 protein. c Endogenous SET8 immunoprecipitation in HeLa cells in presence or absence of the proteasome inhibitor MG132. Western blot (left side) detecting the amount of ADP-ribosylation in GFP (IP: control), SET8 immunoprecipitates (top panel) as well as the amount of SET8 protein (bottom panel). Respective densitometry analyses of SET8 ADP-ribosylation abundance representative of at least 2 biological experiments are shown (right side; n = 2).