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. 1997 Apr 7;185(7):1287–1294. doi: 10.1084/jem.185.7.1287

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The ICE inhibitory activity of human vascular SMC is heatlabile and expresses a molecular size of 50–100 kD. (A) Heat treatment reverses the inhibitory activity of the ICE inhibitor. Recombinant IL-1β precursor (10 μg/ml, 15 μl) was incubated (37°C, 15 min) with heattreated (Heated; 70°C, 30 min) or untreated supernatants (15 μl). Immediately thereafter, recombinant ICE_P30 (0.4 μg/ml, 15 μl) was added. All samples were separated by SDS-PAGE and analyzed by Western blot with the IL-1β–specific antibody (Fib 3, 1:1,000). Similar results were obtained in two independent experiments. (B) Ultrafiltration of inhibitory SMC supernatants. The supernatant of SMC was filtered through 0.2 μm filters and a 100-kD cutoff filter. This preparation was applied to 50-kD cutoff ultrafiltration (50 conc). The run-through (50 run) of this preparation was then filtered through a 30-kD cutoff filter and the concentrate was collected (30 conc). These samples (15 μl) were tested in the processing assay as described above. Three similar experiments were performed.