Targeted disruption of the Pak1 allele. (A) Partial restriction map of the native Pak1 gene (genomic locus), the targeting vector replacing the coding sequence of a portion of the N-terminus, including the p21-binding domain (PBD) and the inhibitory domain (ID) with the Neo-resistance gene in the antisense orientation (targeting vector), and the organization of the targeted Pak1 allele (targeted allele). The 1-kb genomic probe used for screening is indicated along with the expected sizes of the wild-type (WT) and targeted HindIII fragments. (B) Genomic Southern blot analysis (left panel). The nontargeted Pak1 allele (10 kb) is visualized in the WT (+/+) mice and the targeted allele (8 kb) is visualized in the Pak1^−/− (−/−) mice. Both bands can be appreciated in the heterozygous (+/−) mice. Western blot analysis (center panel). WT and Pak1^−/− bone marrow–derived mast cell (BMMC) lysates were subjected to immunoblotting with anti-Pak1. The 68-kDa Pak1 protein is present in WT BMMCs and absent in the Pak1^−/− cells. RT-PCR analysis (right panel). Pak1 cDNA was amplified by PCR from BMMCs to generate a 352–base pair fragment (corresponding to base pairs 306-658) in the WT cells, which is absent in the Pak1^−/− cells. GAPDH mRNA in WT and Pak1^−/− BMMCs is also shown.