Pak1 is a critical mediator of mast cell degranulation in vitro. Mast cell degranulation was assessed by measuring the release of β-hexosaminidase. (A) IgE-primed WT and Pak1^−/− BMMCs were stimulated with DNP-HSA for 15 minutes. To determine FcϵRI-independent degranulation, cells were alternatively stimulated with calcimycin (A23187). In all conditions, β-hexosaminidase activity was measured in the supernatant and the extent of degranulation is reported as a percentage of total cellular β-hexosaminidase activity. Data are means plus or minus SEM from triplicate samples in 4 independent experiments. *P < .05, WT versus Pak1^−/−, unpaired, 2-tailed, Student t test. (B) Confocal laser scanning microscopy images of CD63-EGFP in BMMCs. CD63-EGFP fusion protein was introduced into WT and Pak1^−/− progenitors by retroviral transduction as described in “Methods.” Incorporation of the expressed CD63-EGFP into secretory vesicle membranes allows visualization of vesicle location. Fluorescence images of CD63-EGFP (left) and DAPI (right) are shown. Images are representative of 3 independent experiments. (C) Wild-type Pak1 was reintroduced into Pak1^−/− BMMCs by lentiviral transduction. Expression of Pak1 in transduced Wt and Pak1^−/− BMMCs. The recombinant and endogenous Pak1 proteins are indicated. (D) Release of β-hexosaminidase was measured after sensitization and antigen stimulation as in panel A. Data are expressed as a percentage of WT degranulation. Proof of phenotypic rescue by reintroduction of Pak1 is shown for a single transduced mast cell line assayed in triplicate.