Pak1 is required for normal allergen-induced cytoskeletal changes and vesicle trafficking. In all panels, cells were imaged at baseline (NS), after IgE-priming (4 hours), or following IgE-priming and DNP-HSA stimulation (5 minutes) as indicated. (A) WT and Pak1^−/− BMMCs were fixed and stained with Alexa-488 phalloidin (to detect F-actin intensity) and DAPI nuclear stain. The images are representative of 5 experiments. (B) The number of cells showing F-actin disassembly was estimated under indicated conditions using a quantitative intensity analysis of Image J. Data are expressed as a percentage of cells with fragmented rings (= number of cells with fragmented ring/total number of cells imaged × 100) for 4 independent experiments (100 cells counted per experiment); *P < .001, WT versus Pak1^−/−, unpaired, 2-tailed, Student t test. (C) Confocal laser scanning microscopy images of WT or Pak1^−/− BMMCs transduced with pCL1EGFP (control, “−”) or pCL1EGFP-PAK1 (Pak1 transgene, “+”) as indicated. Imaged cells have been treated with IgE and DNP.