Fig. 6. Negative regulatory activity of A20 on the NF-B activation mediated by PKC. (A) HEK293 and Jurkat cells were left untreated or treated with PMA (40 ng/ml) plus ionomycin (1 µM) or TNF (5 ng/ml) for 6 hours. Lysates (80 µg) extracted from treated and untreated cells were separated by SDS-PAGE, blotted onto nitrocellulose membranes and analyzed by immunoblotting (WB) to monitor A20 protein expression. (B) HEK293 cells were transfected with an empty plasmid or plasmid encoding a shRNA against human A20. 24 hours later, the expression level of A20 was monitored by western blot experiments in cells left untreated or stimulated with PMA (40 ng/ml) plus ionomycin (1 µM) for 6 hours. (C,D) HEK293 cells were transiently cotransfected with the indicated plasmid DNA, together with NF-B–luciferase and β-galactosidase reporter vectors. 16 hours after transfection, cell lysates were prepared and luciferase activity was measured. As a control, a plasmid expressing a shRNA validated to target expression of eGFP was used (shCTR). The data shown represent the relative luciferase activity normalized against β-galactosidase activity and are representative of five independent experiments performed in triplicate. (E) HEK293 cells were transiently cotransfected with the indicated plasmid DNA, together with NF-B–luciferase and β-galactosidase reporter vectors. 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 represent the relative luciferase activity normalized against β-galactosidase activity and are representative of five independent experiments performed in triplicate.