Fig. 3

ARH3 regulates basal and hydrogen peroxide-induced serine ADP-ribosylation in vivo. a Venn diagrams of unique ADP-ribosylated peptides of wild type (WT) and ARH3 KO MEF cells under basal and H₂O₂-treated conditions. b Unique ADP-ribosylation sites detected by EThcD fragmentation in the different samples. c Gene ontology analysis of the identified ADPr-modified proteins using the PANTHER database. Shown on the left are the P-values and on the right the number of identified and annotated ADP-ribosylated proteins. d Validation of mono-ARH activity of ARH3 on the nuclear protein HMGB1. Recombinant HMGB1 was in vitro ADP-ribosylated using recombinant ARTD1 in the presence of ³²P-labeled NAD⁺. Equal fractions were left untreated (Input) or were treated with PARG or ARH3. Above: radioactivity exposure, below: Coomassie Blue-stained poly-acrylamide gel. e Motif searches for ADP-ribosylated peptides with a mascot site localization score >80% in MEF cells using Weblogo. f Energy minimized binding mode of an acetyl-KSG peptide with ADPr-Ser modification. The surface of ARH3 (including the binding-site magnesium ions) is colored according to electrostatic potential (on a scale of −5 to 5 kT/e). The positively charged amino group of the K side chain and the backbone amide groups point toward the region of the surface with negative potential