Fig. 3. A20 inhibits PKC-mediated NF-B activation. (A) HEK293 cells were transfected with empty vector or with the indicated expression vectors, together with an NF-B–luciferase reporter plasmid. 24 hours later, cells were left untreated or treated with PMA (40 ng/ml) plus ionomycin (1 µM) for 5 hours and luciferase activity was measured. The data shown are representative of six independent experiments. (B) HEK293 cells were transfected with empty vector or with the indicated expression plasmids. 24 hours later, cells were treated with PMA (40 ng/ml) plus ionomycin (1 µM) for 40 minutes. 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. (C) HEK293 cells, transfected with an empty expression vector or vector encoding A20wt or A20 mut, were treated with PMA (40 ng/ml) plus ionomycin (1 µM) for the indicated periods of time. Cell lysates were then prepared and analyzed for degradation of the inhibitory subunit I-B by immunoblotting (WP).