FIGURE 5.

Reduced NF-κB activation and IL-6 production in the S132W mutant of CD154. The S132W mutant has substantially reduced activity in NF-κB activation and IL-6 secretion assays. A, HEK293T cells stably transfected with CD40 were transiently transfected with NF-κB reporter and incubated with or without CD154 WT or mutant proteins (5∼20 μg/ml) for 24 h. The cells were then subjected to the luciferase assay. B, human primary B cells were prepared as described in “Experimental Procedures,” incubated with or without CD154 wild type or mutant proteins for 7 days. IL-6 protein secreted into supernatants was measured by ELISA. C, THP-1 cells were treated with 10 μg/ml wild type or the indicated mutant forms of CD154 for 12 h and then subjected to semiquantitative RT-PCR analysis (left). Relative intensities of the ethidium bromide stained bands were analyzed by TINA2.0 (Strauben, Hardt, Germany) (right). Data are the means ± S.E. of three independent experiments.