Fig. 2. The deubiquitylation activity of A20 inhibits interaction of BCL10-IKK/NEMO in mammalian cells. (A) Lysates from HEK293 cells transfected with the indicated expression vectors were immunoprecipitated with mAb against IKK/NEMO. Immunocomplexes were separated by SDS-PAGE, transferred onto membranes and subsequently probed with antisera specific for BCL10 and IKK/NEMO. A fraction of the transfection reaction mixture was separated by SDS-PAGE, blotted onto nitrocellulose membranes and analyzed by immunoblotting to visualize protein expression. The results shown are representative of five separate experiments. (B,C) HEK293 cells were transiently cotransfected with an expression vector encoding the indicated polypeptides, together with NF-B–luciferase and β-galactosidase reporter vectors. The total amount of transfected plasmid DNA was maintained constant by adding empty vector. 16 hours after transfection, cell lysates were prepared and luciferase activity was measured. The data shown represent the relative luciferase activity normalized against β-galactosidase activity and are representative of six independent experiments performed in triplicate. Right panels: a fraction of the transfection reaction mixture was separated by SDS-PAGE, blotted onto nitrocellulose membranes and analyzed by immunoblotting to monitor protein expression. (D,E) Lysates from HEK293 cells transfected with the indicated expression vectors were immunoprecipitated with mAb against IKK/NEMO. Immunocomplexes were separated by SDS-PAGE, transferred onto membranes and subsequently probed with antibodies against ubiquitin and IKK/NEMO. The results shown are representative of four separate experiments.