FIGURE 5.

Simultaneous ligation of CD154 to CD40 and α5β1 on CD40-transfected U937 cells modulates CD154-induced MAPK signaling. A, U937 cells were transfected with an empty vector (U937/vector) or with CD40WT (U937/CD40WT) and analyzed for cell surface expression of CD40 and α5β1 by flow cytometry. Cells were analyzed using specific anti-CD40 mAbs (G28.5) or anti-α5β1 mAbs (JBS5). B, vector (vect)-transfected or CD40-transfected U937 cells were stimulated at 37 °C with CD154 WT (250 ng/sample) for the indicated time points. Medium (Med) was used as a negative control. Cells were lysed, and total cell lysates were analyzed by immunoblot using anti-phospho-p38-specific and anti-phospho-ERK1/2-specific Abs. Anti-p38 and anti-ERK1/2 total antibodies were used to control the sample loading. 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). Results are representative of three independent experiments.