Peptidylarginine Deiminase 4 Activity

Lung digest supernatant was harvested as described above. The PAD4 Inhibitor Screening Assay Kit (AMC) from Cayman Chemical (Ann Arbor, MI, USA) was used to assess PAD4 activity according to the manufacturer's instructions. Briefly, a fluorescent substrate consisting of an arginine, a carboxybenzyl group, and a fluorophore was incubated with the sample. In the absence of active PAD4, the substrate is unaltered and the fluorophore is released upon addition of the developer. In the presence of active PAD4, the substrate is citrullinated preventing the release of the fluorophore upon addition of the developer. Fluorescence was measured in a Clariostar Omega from BMG Labtech (Cary, NC, USA) with excitation set to 360 ± 15 nm and emission set to 450 ± 20 nm. Fluorescent signal is inversely proportional to PAD4 activity. Sample buffer alone was set to 0% activity and recombinant PAD4 enzyme was set to 100% activity. Sample activity was compared to the activity of the recombinant enzyme. Cl-amidine, an irreversible and cell-permeable pan PAD inhibitor, was used at a final concentration of 100 μM per the manufacturer's instructions. Cl-amidine potency is unstable and loses activity very quickly over time (21). For this reason, we used the inhibitor to validate specificity of the assay and not in a function blocking capacity.