The recruitment of pY and pBtk were reduced in WAS memory B cells. (A-D) TIRFM and IRM analysis of pY and pBtk staining in the contact zone of HCs and WAS (P1-12) naive and memory B cells incubated with membrane-tethered Fab′–anti-Ig. Shown are representative images (A-D) and the MFI (±SD) of pY (E) and pBtk (F) in the B-cell contact zone from 3 independent experiments. Bars, 2.5 µm. *P < .01, compared with WAS naive B cells. Enriched B cells from HCs or WAS (P5-15) PBMCs were incubated with soluble antigens for varying lengths of time, fixed, permeabilized, and then stained with antiphosphorylated Erk (G) and Btk (H) antibody. The colocalization coefficients between BCR and pY and pBtk staining were determined using NIS-Elements AR 3.2 software. Shown are the colocalization coefficients (±SD) (I) from ∼50 individual cells of 3 independent experiments. Bars, 2.5 µm. *P < .01, compared with WAS naive or memory B cells.