NF-κB activity induced by BCR ligation is impaired in the absence of Vav proteins. (A) Splenocytes from wild-type and Vav-deficient mice (3 mice per group) were incubated with or without 10 μg/mL Fab₂ α-IgM for 16 hours. Subsequently, mature B cells were identified as HSA^low, CD21⁺, or CD23⁺, and c-Rel levels were analyzed by flow cytometry. (□) Cells incubated in culture medium. (▪) Stimulated cells. (B) B cells from wild-type mice (□) or Vav-deficient (▪) were stimulated as in panel A. Subsequently, nuclear extracts were prepared, and c-Rel nuclear complexes were measured and represented as indicated in Figure 3D. (C) Splenic B cells from wild-type, Vav-1-, Vav-2-, or Vav-1/2-deficient mice were stimulated with 10 μg/mL Fab₂ α-IgM for the indicated time points. Phosphorylation status of IκBα (p-IκBα) was analyzed by Western blot. Stripping and reprobing with α-IκBα antibodies was used as loading control. Error bars correspond to standard deviation.