A Pak/MAPK pathway regulates Nf1 haploinsufficient mast cell hyperproliferation. (A) Mast cells were starved overnight in RPMI and plated in a 24-well plate at 3 × 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 for 72 hours, and viable cells were measured by trypan blue exclusion. Results are expressed as percentage of input number of cells at 72 hours after stimulation. Each value represents the mean, and the 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-D) Mast cells were serum starved overnight, stimulated with SCF, and cell lysates isolated at 0 and 2 minutes after stimulation. A total of 100 μg of protein was used for each time point. Levels of active Erk1 (B) and Mek1 (C,D) were determined by Western blotting using phospho-specific antibodies. Levels of total Erk1 and Mek1 are shown as loading controls. Western blot of the results is shown and is a representative of 3 independent experiments. Vertical lines in panel D have been inserted to indicate repositioned gel lanes for consistency with other blots.