Fig 2. Caspase-1 activity in cell-extract decreases over time.

A) Rapid cleavage of caspase-1 and IL-18 into their mature forms during incubation of cell extract from 3x10⁸ THP1 cells/ml at 37°C. ASC was used as a loading control. B) Cell-extracts were pre-incubated at 37°C for various time points and then loaded simultaneously into the fluorimeter and caspase-1 activity (measured as maximal slope: AFU/min) was measured. Dashed line denotes half-life. C) YVAD-cmk (50 μM) inhibits caspase-1 activity and cleavage. Cell-extracts were incubated at 4 and 37°C in presence or absence of YVAD-cmk for 1h and WEHD-afc cleavage measured (AFU/min). Non-YVAD-cmk inhibitable baseline activity is indicated below the dashed-line. Immunoblots for caspase-1 activation and cleavage of endogenous IL-18 was used to confirm caspase-1 specific activity. ***: p<0.0001(ANOVA). Representative gels from n = 4 and n = 3 independent experiments (A and C, respectively). Data are expressed as mean ± SEM for n = 4 (B) and n = 3 (C) independent experiments.