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

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ICE inhibitory activity of SMC is membrane associated. The supernatant (SN) of SMC was collected, the cells were trypsinized, washed, and half of the cells was lysed by freeze–thaw cycles (Cell). The other part of the cells was treated by digitonin (0.007%) and centrifuged (20 min, 20,000 g). The supernatant (Dig-SN) and the remaining pellet (Dig-Cell) of this centrifugation were collected. For inhibition assay, these samples were incubated with recombinant IL-1β precursor (10 μg/ml, 15 μl), and subsequently with recombinant ICE_P30 (0.4 μg/ml, 15 μl). The products 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.