The levels of tyrosine and Btk phosphorylation in BCR clusters in response to sAg is reduced in Mst1 DKO B cells. 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 (n = 4). (A-F) After fixation and permeabilization, the cells were stained for pY, pBtk, pCD19, and pErk and analyzed by using confocal microscopy. (G-I) The Pearson’s correlation coefficients between BCR and pY/pBtk and pCD19/pErk staining in sAg-stimulated cells were determined by using NIS-Elements AR 3.2 software. (J-M) Flow cytometry analysis of the MFI of pY, pBtk, pCD19, and pErk after stimulation with sAgs from 3 independent experiments. Shown are representative images at indicated times and the average values (± standard deviation) of ∼50 cells from 3 independent experiments. Scale bars, 2.5 μm. One-way analysis of variance with the Tukey test was used to perform multiple group comparisons; *P < .01.