FIG. 4.
Dominant negative RacN17 inhibits MyD88-, IRAK-1-, and TRAF-6-driven Gal4-p65(1–551) activity. EL4.NOB-1 cells (7 × 106) were transiently transfected with 5 μg of plasmids encoding (A) MyD88, (B) IRAK-1, and (C) TRAF-6 along with 1.25 or 2.5 μg of dominant negative (DN) Rac1 as indicated, 2.5 μg of β-galactosidase, 2.5 μg of Gal4-p65(1–551), and 5 μg of Gal4-luciferase. Following stimulation with IL-1 (10 ng/ml), extracts were prepared and measured for luciferase activity. Results are normalized for β-galactosidase activity and are represented as fold increase over nonstimulated empty vector (EV) control. (D) The effect of cotransfecting EL4.NOB-1 cells with RacV12 and RacN17 on Gal4-p65(1–551) and AU1-tagged MyD88 (in the case of RacN17) expression was determined by Western blotting of lysates from cells which had been transfected with 5 μg of RacV12, RacN17, or AU1-MyD88 and 2.5 μg of Gal4-p65(1–551) as indicated. End., endogenus.