Effect of A20 on TNF-induced activation of initiator caspases 8 and 2. A20 inhibits TNF-induced activation of the initiator caspases 8 and 2 by blocking their proteolytic cleavage. Noninfected PAECs and PAECs infected with rAd.β-gal or rAdA20 were treated with CHX (2 μg/mL) alone or with CHX and TNF (100 U/mL) for 6 hours. Caspase 8 (A) and caspase 2 (B) activities were then analyzed in the PAEC lysates by means of a colorimetric assay based on spectrophotometric detection of the chromophore p-nitroanilide (pNA) after cleavage from the caspase-specific–labeled substrates IETD-ρNA for caspase 8 (panel A) and VDVAD-pNA for caspase 2 (panel B). Expression of A20 significantly inhibited TNF-induced activation of caspases 8 and 2. Western blot analysis of procaspase 8 demonstrated that A20 inhibited TNF-induced proteolytic cleavage of caspase 8 (A). The molecular mass of the inactive proform (57 kDa) of the procaspase 8 is indicated (arrows). Data shown represent mean ± SD of triplicate values and are representative of 4 independent experiments. Light gray bars correspond to NI cells; dark gray bars to rAdA20-infected cells; and medium gray bars to rAdβ-gal–infected cells.