Analyses of NF-κB signaling pathways in B cells of Tg mice. (A) Cytoplasmic (Cyt) and nuclear (Nuc) proteins were isolated from purified B cells of Tg and WT mice and analyzed by immunoblotting with Ab to the indicated proteins. The data are representative of 3 independent experiments. BCL10 was identified using a mAb that recognizes human but not mouse BCL10. (B) B cells purified from the spleens of Tg and WT littermates were stimulated with anti-IgM for the indicated times and harvested for preparation of nuclear extracts. Western blotting was used to assess expression levels of components of the canonical (c-Rel, p50, p65) and noncanonical (RelB) NF-κB signaling pathways. Levels of each protein were normalized to β-actin at time zero for WT cells and are indicated numerically. (C) Western blot analysis of IκBα and β-actin levels in splenic B cells stimulated by BCR ligation for the indicated times. The ratio of IκBα to β-actin levels is indicated for each time point normalized to a value of 1 the ratio for WT spleen cells at time zero. (D) Expression of BAFF in serum. The levels of BAFF in sera of 8- to 10-week-old WT and Tg mice were measured by ELISA. Each data point represents a single mouse.