Vav-deficient mice show defects in MAP kinase, Akt, and IκB activation in response to LPS or anti-CD180 stimulation. B cells were stimulated with (A) anti-CD180 (5 μg/mL for 15 minutes), then analyzed for activation of JNK and ERK. (Top) JNK activity assessed by measuring c-Jun phosphorylation. (Middle) ERK activity assessed by blotting with anti-phospho ERK. (Bottom) Total ERK levels assessed by blotting with anti-ERK1 and -ERK2 antibodies. (B) LPS (10 μg/mL for 20 minutes) was then analyzed for activation of ERK activation using anti-phospho ERK. In this experiment anti-ERK-2 was used as the loading control. (C) 5 μg/mL anti-CD180 or 50 ng/mL PMA (phorbol 12-myristate-13-acetate) for the indicated times or (D) 10 μg/mL LPS for 20 minutes. Akt and IκBα phosphorylation were assessed by Western blot using phosphospecific antibodies. Subsequently, blots were stripped and reprobed with Akt or IκBα antibodies, respectively, to confirm equivalent protein loading.