Ectopic NB1 expression confers PR3 surface binding. Transfection of a NB1 expression plasmid into HEK293 cells resulted in NB1 surface expression (A; bold line for NB1-transfected cells, thin line for ctrl-transfected cells, isotype control staining for ctrl- and NB1-transfection is depicted as dotted lines). Monoclonal anti-PR3 antibodies did not bind to any of these cells as shown for CLB12.8. (B) NB1 transfected (bold line) and mock-transfected cells (thin line) were incubated on ice for 2 hours with 1 μg/mL purified PR3 or equimolar concentrations of neutrophil elastase (HNE) or myeloperoxidase (MPO), washed and stained for surface display. (C) Binding of PR3 was specifically elevated in NB1-transfected cells (▪) as compared with mock-transfected (□) cells (MFI, 163 ± 33 versus 25 ± 7; *P < .05; n = 5 independent transfections). HNE and MPO binding was unaffected by NB1 transfection (MFI: HNE, 36 ± 10 versus 39 ± 16, and MPO, 45 ± 12 versus 39 ± 6; n = 3 independent transfections). Error bars indicate SEM.