Processing of Neutrophil α-Defensins Does Not Rely on Serine Proteases In Vivo
Fig 3
35S-proHNP processing assay using promyelocytic PLB-985 proteases.
(A) PLB-985 cells was subjected to nitrogen cavitation followed by pelleting of the postnuclear supernatant containing cytosol, organelles (including granules), and cell membranes. This pellet (P2) was permeabilized in PBS/1% Triton X-100. Preservation of serine protease activity was verified by measuring elastase/proteinase 3 activity by spectrophotometry following degradation rate of methoxysuccinyl-Ala-Ala-Pro-Val-P-nitroanilide. (B) 35S-proHNP was incubated with P2 from PLB-985 cells, incubated at 37°C for 3 hours, and processing tested by SDS-Tricine-PAGE followed by fluorography. (C) P2 was mixed with the serine proteinase inhibitors DFP, PMSF, or elastase inhibitor IV, mixed with 35S-proHNP, and incubated for 6 hours. Processing was analyzed by SDS-Tricine-PAGE followed by fluorography. Complete inhibition of serine proteases by DFP was verified by protease inhibition assays (data not shown).