Increased migration of Nf1^+/− mast cells is mediated through a Pak/p38 pathway. (A) Mast cells were starved overnight in RPMI without serum and plated in the upper well of a transwell chamber at 10⁵ per well in triplicate samples after treatment with DMSO (control; ■), 10 μM of selective p38 inhibitor SB203580 (), or 10 μM of selective Mek1 inhibitor PD98059 (□). Cells were then stimulated with 25 ng of SCF in the lower chamber for 4 hours, and mast cells that had migrated to the bottom surface of the CH296-coated membrane in response to SCF were counted after staining the cells with crystal violet. Results are expressed as cells per 20× high-power field. Each value represents the mean; error bars represent the SEM of 6 independent experiments. *P < .05 compared with WT control. **P < .05 compared with Nf1^+/− control. #P < .05 compared with DMSO-treated cells within a genotype using Student unpaired t test. (B) Mast cells were serum starved overnight, stimulated with SCF, and cell lysates isolated at 0 and 5 minutes after stimulation. A total of 100 μg of protein was used for each time point. Levels of active p38 were determined by Western blotting using phospho-specific antibodies. Level of total p38 is shown as a loading control. Western blot of the results is shown and is representative of 3 independent experiments.