Fig. 4. TRIP6 is involved in TNF-induced NF-B activation. (A) TRIP6 potentiates TNF-induced NF-B activation. 293 cells (1x10⁵) were transfected with 0.1 µg NF-B luciferase reporter plasmid and the indicated amounts of TRIP6 expression plasmid. 14 hours after transfection, cells were treated with TNF (10 ng ml^-1) (filled bars) or left untreated (empty bars) for 6 hours before luciferase assays were performed. (B) TRIP6 has no significant effect on IFN--induced IRF-1 activation. 293 cells (1x10⁵) were transfected with 0.1 µg IRF-1 luciferase reporter plasmid and 1 µg TRIP6-encoding or control plasmid. 14 hours after transfection, cells were treated with IFN- (100 ng ml^-1) (filled bars) or left untreated (empty bars) for 6 hours before luciferase assays were performed. (C) TRIP6 mutants inhibit TNF-induced NF-B activation. 293 cells (1x10⁵) were transfected with 0.1 µg NF-B luciferase reporter plasmid and 0.5 µg TRIP6-(280-480)-encoding (filled bars), TRIP-(1-279)-encoding (dashed bars) or empty control (empty bars) plasmid. 14 hours after transfection, cells were treated with TNF (10 ng ml^-1) or left untreated, as indicated, for 6 hours before luciferase assays were performed. (D) TRIP6 mutants inhibit TRAF2- and TRADD-induced NF-B activation. 293 cells (1x10⁵) were transfected with 0.1 µg NF-B luciferase reporter plasmid, 0.5 µg HA-tagged TRIP6-(280-480)-encoding (filled bars), HA-tagged TRIP6-(1-279)-encoding (dashed bars) or empty control (empty bars) plasmid, and 0.5 µg of Flag-tagged TRAF2 or TRADD expression plasmid, as indicated. Luciferase assays were performed 14 hours after transfection. Expression levels of the transfected proteins were analysed by western blot with anti-Flag and anti-HA antibodies (bottom).