Inhibition of mast cell–and mast cell protease–mediated CTAP-III conversion. Increasing concentrations of purified natural neutrophil CathG and mast cell chymase were incubated with a fixed concentration of 3 μM CTAP-III for 30 minutes at 37°C in processing buffer. Subsequently, 2 μL of each sample was subjected to SDS-PAGE and Western blotting and thereafter stained with Rα-βTG and IRDye 800-conjugated goat α-rabbit antiserum. The amount of generated NAP-2 is shown as assessed by Li-cor quantification against a standard of NAP-2 run in parallel. Data represent mean ± SD from 3 independent experiments (A). Likewise, fixed concentrations of CathG (500 ng/mL) (B) and chymase (250 ng/mL) (C) were incubated with 3 μM CTAP-III under the same conditions in the absence or presence of inhibitors SBTI (1 μg/mL), aprotinin (1 μg/mL), or PMSF (2 mM) for 30 minutes. Correspondingly, human skin MCs (1 × 10⁴/mL) prestimulated with 3 μg/mL α-IgE for 30 minutes were incubated with 3 μM CTAP-III in the absence or presence of inhibitors as indicated above (D). The Western blots shown in panels B-D were performed and developed using the primary and secondary antisera given in Figure 1. The migration of untreated CTAP-III (CT) and NAP-2 (NA) is indicated. For quantification of formed NAP-2, fluorescence of NAP-2-bands was determined by Li-cor analysis and compared with that of 50 ng standardized NAP-2 on the same blot. One representative experiment of 3 is shown.