Figure 3

Effects of N17Rac1, DNTRAF 2, 3, or 6 on CD40-induced ROS production. The cells were transiently transfected with either empty vector or one of the DN expression plasmids, along with a red fluorescent protein, DsRed. Intracellular ROS production after a 20-min stimulation with anti-CD40 was measured (described in Figure 1). (A) Changes in the fluorescence to yellow, due to the green fluorescence from CD40-induced ROS production, are clearly shown in cells that were doubly transfected with the vector and DsRed plasmids. In the case of the concurrent expression of DsRed and a DN mutant of Rac1 (N17Rac1), however, not all of the cells responded to the CD40 stimulation that led to fluorescence changes. The fluorescence intensity after CD40 ligation relative to the control is shown in cells that were transfected either with a vector or a DN mutant of Rac1 (N17Rac1). The average fluorescence intensity from at least 50 cells from three experiments are presented as the average ± SD; ^*, P < 0.001. (B) The relative fluorescence intensity from ROS production after CD40 ligation for 20 min in cells that were transfected with either vector alone or with each DNTRAF is given as a ratio to control fluorescence. Data obtained as in (A) are presented as the average ± S.D.