B cell receptor (BCR)-induced serine/threonine kinase (Akt) activation is inhibited in Rictor knockout (KO) B cells.

To mimic soluble antigen (sAg), splenic wild-type (WT) or Rictor KO B cells were incubated with Alexa Fluor (AF) 546–monobiotinylated (mB)-Fab′–anti-immunoglobulin (Ig) for 10 min at 4°C to label the BCR. Then, the cells were either incubated with streptavidin or with the medium alone (0 min) as a control at 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for phosphorylated Rictor (pRictor) (A) or phosphorylated Akt (pAkt) (D and E) and analyzed using confocal microscopy (CFm). The mean fluorescence intensity (MFI) of pRictor (C) or pAkt (F) and the correlation coefficients (B and H) between the BCR and pRictor or pAkt were quantified using NIS-Elements AR 3.2 software. Flow cytometry analysis of MFI of pAkt after stimulation with sAg (G). Shown are representative images and mean values (±SD) from 3 independent experiments, in which over 50 cells were individually analyzed using NIS-Elements AR 3.2 software. Scale bars, 2.5 μm. Mann–Whitney U test was used to do the statistics, * p < 0.01. The numerical data (for B, C, E, F, G, and H) can be found in S1 Data.