The levels of tyrosine and Btk phosphorylation in BCR clusters in response to sAg is reduced in Dock8 KO B cells. Murine splenic B cells were incubated with AF546–mB-Fab′–anti-Ig without or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for pY, pBtk, Dock8, WASP, and pWASP and were analyzed using CFm (A-B,F) or flow cytometry (D-E,I). The Pearson’s correlation coefficients between BCR and pY/pBtk (C) or between BCR and Dock8/pWASP (G) staining in sAg-stimulated cells were determined using NIS-Elements AR 3.2 software. (H) Real-time PCR to examine the mRNA expression levels of wasp in sorted Dock8 B cells. Flow cytometry analysis of the MFI of pY and pBtk in splenic B cells after stimulation with sAgs (D-E) or the MFI of WASP in splenic B cells without stimulation (I). Shown are representative images in which more than 50 cells were individually analyzed using NIS-Elements AR 3.2 software, and mean values (±SD) are from 3 independent experiments. Scale bars, 2.5 μm. *P < .01; **P < .001. ISO, isotype control.