The levels of pWASP are increased in Mst1 KO B cells via promoting the expression of WIP. 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). After fixation and permeabilization, the cells were stained for pWASP and WIP and analyzed by using confocal microscopy (A-B and G-H). The Pearson’s correlation coefficients between BCR and pWASP staining in sAg-stimulated cells were determined by using NIS-Elements AR 3.2 software (C). The levels of pWASP in the cytoplasm and membrane of B cells stimulated with sAg at 5 minutes were quantified by using NIS-Elements AR 3.2 software (D). Flow cytometry or western blot analysis of the WASP and pWASP expression of B cells stimulated with sAg with varying lengths of time (E-F). (I) Pearson’s correlation coefficients between BCR and WIP staining in sAg-stimulated cells were determined by using NIS-Elements AR 3.2 software. (J) Western blot analysis of WIP in WT and Mst1 KO B cells. (K) Real-time polymerase chain reaction analysis of wasp and wip in unstimulated WT and Mst1 KO B cells; the mRNA expression level of wasp and wip from different samples was standardized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Shown are the results from 3 independent experiments and 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, **P < .001.