Figure 1.

The human serpin PI-9 inhibits native and recombinant caspase-1 processing activity. (A) Human recombinant PI-9 (100 nM) was either preincubated for 30 min (−30) with native, monocyte-derived (Mφ-Lysates; equivalent to 10⁶ Mφ/ml) or recombinant (rec.) caspase-1 (15 nM), applied simultaneously (0), or added 30 min after incubation (+30) of proIL-1β (20 nM) with the enzyme for 30 min at 37°C in a final volume of 50 μl processing buffer. (B) Human recombinant caspase-1 (15 nM, top) or human recombinant IL-1β precursor (20 nM, bottom) was preincubated for 30 min with the indicated concentrations of PI-9, before application to the processing assay. Processing was stopped by heating the samples in 10 μl SDS-PAGE sample buffer. The preparations were analyzed by 15% SDS-PAGE and subsequent Western blot analysis using anti–human IL-1β. For control purposes, recombinant mature (mIL-1β, 20 nM) and precursor (pIL-1β, 20 nM) IL-1β were applied. The positions of the molecular weight markers are indicated on the left (in kD). Similar data were obtained in three (A) or five (B) independent experiments.