Effect of SB202190 and D609 on p65 activity and of SB202190 on TNFα- and NiCl2-induced function of p300.
SB202190 and D609 do not inhibit modulation of p65 activity, but SB202190 blocks TNFα- and NiCl2-induced function of p300. (A-B) HUVECs were transiently transfected with plasmids expressing luciferase under control of a 5xGal4 binding site–containing promoter (A, lanes 1 and 2) or were cotransfected with a vector expressing a Gal4-p65 fusion protein (A, lanes 3 and 4, and B). (C) HUVECs were transiently transfected with 1 μg MCP-1 ENH promoter or were cotransfected with 0.5 μg CMVp300 (lanes 3 through 6 and 9 through 12). In all experiments, 36 hours after transfection, cells were either left untreated or stimulated with 2 ng/mL TNFα (lanes 2, 4, 6) or 1.5 mM NiCl2 (lanes 8, 10, 12) in the presence or absence of 20 μM SB202190. Equal transfection and expression efficiencies were controlled by coexpression of a CMV-driven β-Gal reporter gene construct (data not shown). (D) Experiments were performed as in panel C, but SB202190 was replaced by 10 μg/mL D609. Relative luciferase activity is expressed as fold stimulation. In panel B, Gal4-promoter transactivation by expression of Gal4p65 in the absence of TNFα is arbitrarily set as 1, and fold activation of TNFα-induced activity is calculated. Mean values ± SEM from up to 8 independent transfections are shown.
