Characterization of Pak1^−/− bone marrow–derived mast cells. (A) Bone marrow–derived mast cell (BMMC) receptor expression. BMMCs were maintained in culture for 5 weeks, and expression of c-kit and FcϵRI was measured by incubation with antimouse CD 117 (c-kit) PE-conjugated antibody, and anti-DNP monoclonal antibody IgE clone SPE-7 followed by incubation with FITC-conjugated anti–mouse IgE secondary antibody. Double-positive cells (top right quadrant) are mature mast cells, expressing both c-kit and FcϵRI. Data shown are representative of 6 independent lines from each genotype. (Mean WT = 96.1 + 2.3 SEM % vs Pak1^−/− = 95.3 + 1.7 SEM % double-positive cells, n = 6.) (B) IgE-mediated Pak1 activation in BMMCs (representative of 3 independent experiments). IgE-primed BMMCs were stimulated with antigen (DNP) for the indicated times, lysates were precipitated with anti-Pak1 antibody, and Pak1 activity was assayed. (C) Pak1 activation of pS298-MEK1 in Wt and Pak1^−/− BMMCs. IgE-primed BMMCs were stimulated with antigen (DNP) for the indicated times. Cell lysates (“Western blotting”) were subjected to immunoblotting with anti–phospho-S298 MEK1 (top blot) or anti–total MEK1/2 (bottom blot). (D) Effect of IPA-3 treatment in Wt BMMCs. The length of activation and the addition of inhibitor are indicated.