FIGURE 3.

Binding of CD154 to α5β1 is independent of residues Tyr-145, Arg-203, and Asp-117. BJAB or U937 cells were incubated with rsCD154 (250 ng/sample) and wild type or mutant (hCD154 D117E, hCD154 R203A, hCD154 Y145A, and hCD154 R203A/Y145A) for 30 min at 4 °C for BJAB cells and 30 min at 37 °C for U937 cells. Washed cells were then incubated with biotinylated anti-CD154 polyclonal Ab for 30 min at 4 °C, followed by streptavidin-phycoerythrin for 30 min and washed and analyzed by flow cytometry. Filled black plots indicate the cells incubated with streptavidin-phycoerythrin (negative control), as compared with the empty gray plots that represent the binding of rsCD154 to BJAB and U937 cells. Values within plots represent the mean fluorescence intensity for rsCD154 binding (wild type or mutants). Middle plots show the expression of the recombinant competitors. Proteins (2 μg total) were resolved on 12% SDS-polyacrylamide gels and stained with Coomassie Blue (R-250). All results shown are representative of four independent experiments.