Vav regulates Rac2 activation in response to LPS, and Vav GEF activity is required for LPS-induced oxidative burst. (A) Vav^null and WT BMDMs were stimulated with LPS (10 μg/mL) for the indicated time points. Rac2 activation was assayed by pull-down of GTP-Rac2 from cell lysates with PBD-GST fusion protein, followed by Western blotting with antibodies specific to Rac2. Total cell lysates (TCLs) were analyzed for Rac2 content by Western blotting. Densitometry was performed to quantitate GTP-bound Rac2 normalized to total Rac2. (B) Vav^null BMDMs were reconstituted by retroviral transduction with WT Vav1 (Vav1^WT), a GEF-inactive Vav1 (Vav1^GEF-MUT), or GFP control vector. Cells were stimulated with LPS (10 μg/mL), and oxidative burst was determined by lucigenin chemiluminescence. Data represent the mean ± SEM RLU measured at 15 minutes for triplicate samples. Results are representative of 3 independent experiments.