Vav is required for LPS-induced activation of p38 MAPK. (A) BMDMs from WT, Vav^null, and MyD88^−/− mice were stimulated with LPS (10 μg/mL) for the indicated time points. Activation of p38 MAPK was assayed by probing of immunoblots with antibodies recognizing phosphorylated p38 (p-p38). Protein loading was verified by stripping and reprobing of blots with antibodies against total p38. (B) BMDMs from WT and Vav^null mice were stimulated with LPS (0.1 μg/mL), and activation of p38 MAPK was assayed as in panel A. (C) Unmanipulated WT and Vav^null BMDMs were treated with 1 μM H₂O₂ for the indicated time points, and p38 MAPK activation was assayed as in panel A. (D) BMDMs from WT and Vav^null mice were stimulated with LPS (10 μg/mL) and analyzed for activated ASK1 by Western blotting, with phosphospecific antibodies recognizing threonine residue 845 in murine ASK1 (pASK1). Equal protein loading was demonstrated by stripping and reprobing blots with anti-ERK2. Densitometry was performed in all panels to quantitate the indicated phosphorylated protein relative to total levels of the protein. Data are representative of 3 experiments.