Vav is not required for COX2 or iNOS induction in response to LPS stimulation. (A) WT and Vav^null BMDMs were stimulated with LPS (10 μg/mL) and lysed at the indicated time points. Lysates were subsequently analyzed by Western blotting for COX2. Protein loading was verified by stripping and reprobing blots with antibodies against total ERK2. (B) WT and Vav^null BMDMs were stimulated with LPS (10 μg/mL), LPS and IFN-γ (50 U/mL), or LPS and PMA (50 ng/mL) for the indicated time points (18 hours, unless otherwise indicated). Cells were immediately lysed and analyzed by Western blotting for iNOS. Protein loading was verified by stripping and reprobing of blots with antibodies against total Erk-2. Densitometry was performed to quantitate iNOS and COX2 protein levels relative to total Erk-2. (C) BMDMs were stimulated with LPS (10 μg/mL) or LPS and IFN-γ (50 U/mL) for 18 hours. RNI production was analyzed by measuring nitrite content in culture supernatant using the Greiss reagent. For RNI experiments, data shown are mean ± SD of triplicate samples and are representative of 2 independent experiments. COX2 and iNOS expression was examined in more than independent experiments.