FIGURE 6.

BCR activation induces Btk-dependent phosphorylation of Vav and recruitment of phosphorylated Vav to the BCR. A, The surface BCR of splenic B cells from wt (a–i) and xid mice (j–r) were labeled with Cy3-Fab-anti-μ and activated with F(ab′)₂-anti-Ig for varying lengths of time. The cells were fixed, permeabilized, and stained with an Ab specific for phosphorylated Vav at Y174 (pVav). Images were acquired using a confocal fluorescence microscope. Shown are representative images from three independent experiments. Bar, 3 μm. B, The wt splenic B cells that were treated or untreated with LFM A-13 and xid splenic B cells were activated with F(ab′)₂-anti-Ig for varying lengths of time. The cells were fixed, permeabilized, and stained with an Ab specific for pVav Y174. The MFI of pVav was quantified using flow cytometry. Shown is a representative plot of pVav MFI vs the time from three independent experiments. C–F, A20 B cells that were treated with LFM A-13 or left untreated (C and D)as well as splenic wt and xid B cells (E and F) were stimulated with F(ab′)₂-anti-Ig for indicated times and lysed. The lysates were analyzed by SDS-PAGE and Western blot, and probed for pVav Y174. The blots were stripped and reprobed for tubulin as loading controls. The blots were analyzed using densitometry. pVav levels were normalized against tubulin levels, presented as fold increases over unstimulated B cells, and plotted as a function of time. Shown are representative blots and average pVav levels from three independent experiments.