FIGURE 4.

CD154 mutants (R203A and R203A/Y145A) induce p38 MAPK phosphorylation in U937 cell, but not in CD40⁺ BJAB cells. BJAB (A) or U937 (B) cell lines were stimulated at 37 °C with 250 ng of sCD154WT or each of its mutants for the indicated time points. Cell lysates were separated by SDS-PAGE and analyzed by immunoblot using an anti-phospho-p38-specific antibody. Membranes were stripped and re-hybridized with an anti-p38 specific antibody. Medium (Med) was used as negative control. The values below the blots represent arbitrary units of increase in phosphorylation relative to total protein levels, as measured by densitometry using the Quantity One analysis software (Bio-Rad). Blots are representative of three independent experiments.