The recruitment of phosphorylated SH2-containing inositol phosphatase (pSHIP) to B cell receptor (BCR) clusters in B cells stimulated by soluble antigen (sAg) is increased in Rictor knockout (KO) B cells.

Splenic B cells were incubated with Alexa Fluor (AF)546–monobiotinylated (mB)-Fab′–anti- immunoglobulin (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 pSHIP and analyzed using confocal microscopy (CFm) or flow cytometry (A, B and D). The mean fluorescence intensity (MFI) of pSHIP was generated using NIS-Elements AR 3.2 software (C). Flow cytometry analysis of the MFI of pSHIP after stimulation with sAgs (D). The Pearson’s correlation coefficients between BCR and pSHIP staining in sAg-stimulated cells were determined using NIS-Elements AR 3.2 software (E). Shown are representative images at indicated times and the average values (±SD) of about 50 cells from 3 independent experiments. Scale bars, 2.5 μm. Mann–Whitney U test was used to do the statistics, *p < 0.01. The numerical data (for C, D, and E) can be found in S1 Data.