PI3K activity is essential for maintenance of c-Rel expression. (A) PI3K-independent activation of ERK. Splenic B cells obtained from p85α−/− (p85α−/−) or control (p85α+/−) mice on a BALB/c background were stimulated with or without 20 μg/mL anti-IgM F(ab′)2 (anti-μ) at 37°C for the indicated times. BCR-induced activation was evaluated by immunoblotting with a specific antibody against phospho-ERK (p-ERK). The membrane was reblotted with an anti-ERK2 antibody (ERK2). (B) PI3K-independent degradation of IκBα. Splenic B cells were stimulated as in panel A. The degradation of IκBα was evaluated by immunoblotting with an anti-IκBα antibody. The membrane was reblotted with an anti-ERK2 antibody. IκBα levels were normalized by ERK2 levels and indicated as percentage relative to that of the unstimulated lysate (ratio). (C) The whole-cell lysates of splenic B cells obtained from p85α−/− (−/−) or control (+/−) mice on a BALB/c background were subjected to immunoblot analysis with antibodies against c-Rel, RelA, and ERK2. (D) PI3K-dependent maintenance of c-Rel expression. Splenic B cells of C57BL/6 mice were cultured at 5 × 106 cells/mL in the presence or absence of either 100 nM wortmannin (WN) or 10 μg/mL rapamycin (Rap) for the indicated times, and evaluated for the expression level of c-Rel by immunoblotting with an anti–c-Rel antibody. Rapamycin, a potent inhibitor for mTOR, was chosen as a control since wortmannin is known to suppress mTOR activity besides PI3K. The membrane was reblotted with an anti-ERK2 antibody. Data in panels A-D are representative of 3 independent experiments with similar results.
