Pak1 and p38 cooperate to regulate activation and organization of the F-actin cytoskeleton. Mast cells were starved overnight in RPMI and plated in the upper well of a transwell chamber at 10⁵ per well in triplicate samples after treatment with DMSO or 10 μM of selective p38 inhibitor SB203580. Cells were then stimulated with 25 ng of SCF in the lower chamber for 30 minutes, and mast cells were removed from the upper chamber for phalloidin staining of the F-actin cytoskeleton. (A-L) Representative micrographs of phalloidin-stained mast cells analyzed with the Zeiss UV LSM-510 confocal microscope system equipped with a UV Argon laser (351, 364 nm excitation), a visible Argon laser (458, 488 nm excitation) and two Helium-Neon lasers (543, 633 nm excitation). The microscope was equipped with 4 epifluorescence detectors and 1 transillumination detector. The system was mounted on a Zeiss Axiovert 100 inverted microscope and software for image analysis was Zeiss LSM browser R 4.0 (all Carl Zeiss, Thornwood, NY). Green indicates phalloidin stain; blue, DAPI nuclear stain. Original magnification ×400. (M) Fluorescence intensity of phalloidin-stained mast cells, determined by fluorescence cytometry. Data are expressed as fold increases over WT levels; each value represents the mean, and 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.