Identification and expression of pak4 in zebrafish embryos. (A) Phylogeny of the pak family. Putative protein sequences of the kinase domains of human (h), mouse (m) and zebrafish (z) PAKs were analyzed using ClustalW (http://clustalw.genome.jp/). Results are shown as a dendrogram with branch length indicating divergence. PAK5 proteins were also named as PAK7 in Genbank and EnsEMBL databases. Genbank Accessions or EnsEMBL Gene IDs (Zv9): humanPAK1, NM_001128620; humanPAK2, NM_002577; humanPAK3, NM_001128166; humanPAK4, NM_005884; humanPAK5, NM_020341; humanPAK6, NM_020168; mousePAK1, NM_011035; mousePAK2, NM_177326; mousePAK3, NM_008778; mousePAK4, NM_027470; mousePAK5, NM_172858; mousePAK6, NM_001033254; zfPAK1a, ENSDARG00000042624; zPAK1b, NM_201328; zPAK2a, NM_001002717; zPAK2b, NM_001025456; zPAK4, NM_001002222; zPAK5, NM_212962; zfPAK6a, ENSDARG00000027564; zPAK6b, NM_001162487. Zebrafish PAK4 is indicated in red. (B) Quantitative analysis of zebrafish pak4 expression in development. Quantitative RT-PCR was carried out with total RNA (800 ng) extracted from specific embryonic stages and analyzed for pak4 expression. Maternal pak4 mRNA levels rapidly declined between 30% epiboly and shield stages, then remained approximately constant through 24 hpf. Results shown were collected from three individual experiments. (C) Whole-mount in situ hybridization for pak4. Stages are as indicated. 4/8-cell stage – animal pole view, others are lateral view. Signals at and after 30% epiboly were relatively weak and hence staining of these samples for alkaline phosphatase activity was prolonged. A higher magnification of the tail of the 24 hpf embryo is shown to highlight elevated expression in newly formed somites. (D) Immunoprecipitation/western analysis. Total soluble protein was isolated from embryos at multiple stages, immunoprecipitated and prepared for western blotting as described in Section 2. Equal numbers of embryos were used for each sample, which was confirmed by BCA protein assays. Ponceau S staining of the IgG heavy chain band (IgG) is shown below as a control for immunoprecipitation recovery.