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Fig. 7. A20 inhibits CARMA1 and BCL10 function. (A) Jurkat cells were transfected with an empty vector or vector encoding A20wt and A20mut, together with an NF-B–luciferase reporter plasmid. 24 hours later, cells were treated with PMA (40 ng/ml) plus ionomycin (1 µM) for 5 hours and luciferase activity was measured. The data shown are representative of five separate experiments. The expression level of cotransfected A20wt and A20mut, relative to one experiment, was verified by western blot and is shown in the bottom panel. (B) Jurkat cells were transfected as in panel A and then stimulated for 10 hours with antibodies against CD3 and CD28 (1 µg/ml of anti-CD3 and 2 µg/ml of anti-CD28), after which the luciferase activity was determined. The data shown are representative of four independent experiments. (C) Jurkat cells were transfected with empty vector or with an expression plasmid encoding A20. 24 hours later, cells were treated with PMA (40 ng/ml) plus ionomycin (1 µM) for the indicated periods of time. Lysates were then prepared and immunoprecipitated (IP) with mAb against IKK/NEMO. Immunocomplexes were separated by SDS-PAGE, transferred onto membranes and subsequently probed with antibodies (WB) against ubiquitin and IKK/NEMO.

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