Rictor deficiency deregulates the dephosphorylation of ezrin and actin inhibition can rescue the defects of B cell receptor (BCR) signaling.

Splenic B cells were pretreated with or without 0.05 μM Latrunculin B for 30 min and then 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 in the presence of Latrunculin B. (F) Flow cytometry analysis of BCR internalization by quantifying the percentage of biotin-F(ab′)₂–anti-immunoglobulin (Ig)–labeled BCR remaining on the cell surface after the 37°C chase in the presence of Latrunculin B after treatment. Shown are the average percentages (±SD) from 3 independent experiments. *p < 0.01 compared to control B cells. After fixation and permeabilization, the cells were stained for Alexa Fluor 488 (AF488)-phallodin, phosphorylated Ezrin (pEzrin) (A-C), phosphorylated Brutons tyrosine kinase (pBtk), and phosphotyrosine (pY) (G-I) and analyzed using confocal microscopy (CFm) and flow cytometry (D-F and J-K). The Pearson’s correlation coefficients between BCR and phosphorylated SH2-containing inositol phosphatase (pSHIP) staining in soluble antigen (sAg)-stimulated cells were determined using NIS-Elements AR 3.2 software (L). 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. One-way ANOVA with the Tukey test was used to do multiple group comparisons, *p < 0.01. The numerical data (for E, F, J, K, and L) can be found in S1 Data.