Recombinant human histone h3

Chemicals were purchased from Sigma–Aldrich, unless otherwise noted. Electrophoresis reagents were from ThermoFisher Scientific, Gothenburg, Sweden. Recombinant human histone H3.1 was purchased from New England Biolabs (M2503). Expression vectors for the PARP10 catalytic domain constructs N819-V1007 (wild type and mutants) were constructed by sub-cloning synthetic DNA fragments (GeneArt; ThermoFisher Scientific) into pNIC-28 [22 (link)]. The cDNAs encoding human MacroD2^21–245 and human TARG1/C6orf130^1–152 were inserted into pNIC-28 to obtain N-terminal hexahistidine fusions. Human ARH3 expression plasmid (full length sequence in pET26) [23 (link)] was kindly contributed by Friedrich Koch-Nolte (University Medical Center Hamburg-Eppendorf, Hamburg, Germany). All of the proteins were expressed in Escherichia coli strain BL21(DE3)T1R (Sigma–Aldrich, Stockholm, Sweden) and then purified by immobilized metal affinity chromatography, followed by size exclusion chromatography.

Citrullinated histone H3 (H3Cit) ELISA was performed as previously described^{24 ,25} . In brief, 96-well plates were coated with an anti-histone antibody (Cell Death Detection ELISA; Sigma–Aldrich, St Louis, MO, USA) overnight at 4 °C. H3Cit standards (recombinant human peptidylarginine deiminase 4 (PAD4) (Cayman Chemicals, Ann Arbor, MI, USA) and recombinant human histone H3.1 (New England Biolabs, Evry, France)) and plasma samples were incubated at RT for 1.5 h and then washed with PBS containing 0.05% Tween-20. Next, anti-H3Cit antibody (1:1000 ab5103; Abcam, Cambridge, MA, USA) was added for 1.5 h. After another washing step, anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (1:5000 goat anti-rabbit IgG HRP; Bio-Rad Laboratories, Hercules, CA, USA) was incubated for 1 h, washed again and incubated with 3,3’,5,5’ -tetramethylbenzidine (Sigma–Aldrich, St Louis, MO, USA) for 25 min. Finally, the reaction was stopped with 2% sulfuric acid, and absorbance at 450 nm was measured using a Multiskan Spectrum microplate reader (Thermo Scientific Inc., Bremen, Germany).