Attenuated nuclear induction of NF-κB and NF-κB2 processing of Rictor-deficient B cells. (A) Aliquots of B cells or mature B cells purified after tamoxifen pretreatment of Cre-ER^T2Rictor^fl/fl or control mice (as in Figure 3A) were extracted before or after culture with anti-IgM (15 minutes) and then were analyzed by immunoblotting. Shown are signal images for the indicated antibody in 1 of 3 independent experiments with comparable results. (B) Nuclear extracts were prepared from mature B cells of Cre-ER^T2Rictor^fl/fl or control mice pretreated with tamoxifen, and analyzed by EMSA using a consensus NF-κB probe. Shown is the autoradiograph of results from 1 replicate representative of 3 independent experiments: arrowhead indicates complexes formed with NF-κB proteins, and line indicates free probe bands. (C) B cells of Cre-ER^T2Rictor^fl/fl or control mice pretreated with tamoxifen were treated with anti-IgM (1 hour), BAFF (4 hour), or medium, after which nuclear induction of NF-κB was analyzed as in (B). (D) Processing of NF-κB2 from the p100 precursor to p52 form. Western blotting was performed on extracts of B cells of Cre-ER^T2Rictor^fl/fl or control mice pretreated with tamoxifen (as in panel A) after culture (24 hours) with or without BAFF. Shown is a representative result from among 3 independent experiments, along with the mean (± SEM) ratio of p52/p100 calculated after quantitation of p100 and p52 band intensities.