Impaired activation of JNK and NF-κB by BCR in TAK1-deficient B cells. Splenic mature B cells (AA4.1⁻) were isolated from CD19CreTak1^+/+, CD19CreTak1^fl/+, or CD19CreTak1^fl/fl mice. Cells were stimulated with anti-IgM for the indicated times and then lysed. (A) Impaired activation of JNK by BCR in TAK1-deficient B cells. Cell lysates were subjected to direct Western blot analysis with anti–phospho-JNK or anti-JNK antibodies. (B) Normal activation of ERK and p38 by BCR in TAK1-deficient B cells. Cell lysates were subjected to direct Western blot analysis with anti–phospho-ERK, anti–phospho-p38 or anti-p38 antibodies. (C,D) Impaired phosphorylation of IκBα by BCR in TAK1-deficient B cells. Cell lysates were subjected to direct Western blot analysis with anti–phospho-IκBα or anti- IκBα antibodies. (D) Cells were stimulated with anti-IgM for the indicated times in the absence (control) or presence of MG132 before Western blot analysis. (E) Impaired phosphorylation of IKKβ by BCR in TAK1-deficient B cells. Cell lysates were subjected to direct Western blot analysis with anti–phospho-IKKα/β or anti-IKKβ antibodies. (F,G) Impaired activation of NF-κB by BCR in TAK1-deficient B cells. Cell lysates were subjected to NF-κB gel mobility shift analysis. LPS-stimulated mature B cells were used as controls. *A nonspecific band that serves as a loading control. Data are representative of 3 individual experiments.