Fig. 3

DNase-I binds to serum protein(s) but remains active. a Normal human serum (NHS) separated by size using gel filtration was tested for neutrophil extracellular trap (NET) degrading activity (dashed blue line, right y-axis) and primary necrotic chromatin degrading activity (green line, left y-axis). Activity was confined to one peak. Serum with ¹²⁵I-labeled DNase-I was used to determine if degradation corresponded to DNase-I activity. The ¹²⁵I DNase-I incubated with NHS eluted in two peaks as judged by ¹²⁵I radiation (insert) indicating free DNase-I and DNase-I bound to a serum protein(s) or forming multimers. Proteins eluted in both peaks contained NET degradation activity (solid blue line, right y-axis). b AF488-labeled DNase-I was incubated with increasing concentrations of actin, or Gc globulin as negative control, in NHS or heat-inactivated NHS (Hi-NHS) and subjected to native DNA zymogram. At increasing concentrations of actin, DNase-I (green) shifted to a larger/less charged state although it remained active as judged by activity staining (red). a Representative data of two independent experiments. b Representative gel out of three independent experiments. Prim-Nec primary necrotic chromatin, cpm = counts per minute