Activated signaling pathways in Notch2IC-expressing B cells. (A) Decreased levels of pIκBα, IκBα, and p52 in Notch2IC-expressing B cells. Whole-cell extracts from unstimulated cells were subjected to immunoblot analysis with the use of antibodies specific for pIκBα (Ser32/36), IκBα, or an antibody specific for p100/p52. Equal protein loading was controlled by α-tubulin staining. The result is representative for 5 independent experiments. (B-C) The basal activity of the noncanonical NF-κB pathway is decreased in Notch2IC-expressing B cells. Cytoplasmic (CP) and nuclear (N) levels of NF-κB components of splenic B cells from Notch2IC//CD19Cre^+/− and control mice were analyzed by immunoblot with the use of the indicated antibodies. Purity of cytoplasmic and nuclear extracts and equal protein loading were verified by α-tubulin, α-LaminB, and Ponceau staining, respectively. The experiment was performed 4 times. (D-E) In comparison to splenic control B cells, the basal activity of MAPKs and Akt is increased in Notch2IC-expressing B cells. Whole-cell extracts from unstimulated cells were subjected to immunoblot analysis with the use of antibodies specific for pJnk1/2 (Thr183/Tyr185), pErk1/2 (Thr202/Tyr204), pAkt (pS473), and the corresponding nonphosphorylated forms. Equal protein loading was controlled by α-tubulin staining. The result is representative for 5 independent experiments. (F) The phosphorylation of Erk, Jnk, and Akt is higher in wt MZ B cells in comparison to Fo B cells, but it is comparable in Notch2IC-expressing and wt MZ B cells. Splenocytes from Notch2IC//CD19Cre^+/− and control mice were fixed, permeabilized, and intracellularly stained with antibodies specific for the indicated phosphorylated signaling molecules and analyzed by FACS. Histograms show overlays of the abundance of the indicated molecules on lymphocyte-gated, B220⁺ B cells comparing (1) CD21^high (dark gray) and CD21^low (black) control B-cell populations (left), (2) hCD2⁺ Notch2IC//CD19Cre^+/− (black line) and control (gray line) total B cells (middle), or (3) hCD2⁺ Notch2IC//CD19Cre^+/− (black line) and control (gray line) CD21^high B cells (right). Data are representative for 4 independent experiments.