FIGURE 2.

CD40 internalization after CD154 ligation is mediated by the scaffold protein JLP. A, CD40 expression on splenic B lymphocytes with rCD154 stimulation for different times. B lymphocytes from jlp^+/+ or jlp^−/− mice pre-activated with LPS (1 μg/ml) were stimulated with rCD154 (1 μg/ml) for the indicated times and subsequently subjected to CD19 and CD40 staining. CD40 expression on gated CD19⁺ B lymphocytes is shown in the histograms. The percentage MFI reduction of CD40 was calculated by dividing the CD40 MFI at the indicated time points by that at 0 min. B, CD40 expression on splenic B lymphocytes co-cultured with CD154-transfected HEK293 cells. Splenic B lymphocytes from jlp^+/+ or jlp^−/− mice were co-cultured with mock- or CD154-transfected HEK293 cells (HEK293/B cell ratio of 1:50) for 15 min. Cells were harvested and subjected to flow cytometry analysis of CD40 expression. C, CD40 expression on siRNA-transfected WEHI-279 B cells upon rCD154 stimulation. JLP knockdown in WEHI-279 cells with siRNA transfection was confirmed by immunoblot analysis. The percentage MFI reduction of CD40 was calculated by dividing the CD40 MFI after CD154 treatment by that at baseline. Ctrl, control. Total CD40 expression in splenic B lymphocytes at the indicated time points upon soluble CD154 treatment was examined by flow cytometry (D) and immunoblotting (E). For flow cytometry, cells were permeabilized for CD40 staining. The percentage MFI reduction of CD40 expression at the indicated time points is compared with that at 0 min. For immunoblotting, whole-cell lysates were prepared as described above. In the flow cytometry analysis, data from three different experiments were collected and statistically analyzed. In the immunoblot analysis, the indicated band intensity of JLP was normalized by the relevant band intensity of β-actin. Means ± S.D. were collected from at least three independent experiments. Error bars indicate S.D. *, p < 0.05 (Student's t test).