Effect of A20 on TNF-induced activation of effector caspases 3 and 6. A20 inhibits TNF-induced activation of the effector caspases 3 and 6 by blocking their proteolytic cleavage. NI 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 3 (A) and caspase 6 (B) activities were analyzed in the PAEC lysates by means of a colorimetric assay based on spectrophotometric detection of the chromophore ρNA after cleavage from the caspase-specific–labeled substrates DEVD-pNA for caspase 3 (A) and VEID-pNA for caspase 6 (B). Expression of A20 significantly inhibited TNF-induced activation of caspases 3 and 6. Western blot analysis of procaspase 3 demonstrated that A20 inhibited TNF-induced proteolytic cleavage of caspase 3 (A). The molecular mass of the inactive proform of the procaspase 3 (32 kD) is indicated (arrows). Data shown are 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.