By Malcolm Whitman, Amy Sater
Developmental biologists were pushed to enquire development issue signaling in embryos as a way to comprehend the regulatory mechanisms underlying a given developmental strategy. therefore, it truly is serious to discover the technical equipment and experimental designs for development issue signaling in embryos. targeting particular pathways or pathway elements, research of progress issue Signaling in Embryos offers the tools and guidance for experimental layout to check significant elements of phone signaling in vertebrate embryos. The ebook covers a large variety of themes in signaling and numerous present version organisms. part I explores particular signaling pathways or pathway parts. during this part, a few chapters spotlight the biochemistry of signaling pathways in the course of improvement, that is frequently precise from that saw in mobilephone tradition platforms. part II discusses ionic regulatory mechanisms and the 2 chapters in part III research methods of investigating gene law in keeping with extracellular signs. ultimately, part IV addresses rising ideas that facilitate built-in analyses of mobilephone signaling in vivo in embryonic platforms. that includes contributions from specialist researchers, research of progress issue Signaling in Embryos will offer a beginning for extra explorations of the mobile regulatory mechanisms governing vertebrate embryonic improvement.
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Extra resources for Analysis of Growth Factor Signaling in Embryos (Methods in Signal Transduction Series)
9, 967, 2004. 4. Hikasa, H. , The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling, Development, 129, 5227, 2002. 5. Oishi, I. , The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway, Genes Cells, 8, 645, 2003. 6. Lu, W. , Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth, Cell, 119, 97, 2004. 7. C. , Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner, Current Biology, 9, 695, 1999.
A) Measuring GSK-3 kinase activity. Cultured cells or embryos are subjected to the experimental treatment of choice (step 1), collected, and lysed, and the protein-containing fraction is collected (step 2). GSK-3 is immunoprecipitated (step 3), and the isolated GSK-3 is tested against a peptide or full-length protein substrate (step 4) to determine if phosphorylation is occurring (step 5; see text). (b) Testing whether GSK-3 is phosphorylating β-catenin. Embryos are injected at the one- to two-cell stage with mRNA encoding experimental proteins or epitope-tagged β-catenin (step 1).
The underlined sequence represents the position normally phosphorylated by GSK-3. (b) Determine if the protein of interest is phosphorylated. Immunoprecipitated protein is examined for evidence of phosphorylation, for example, by looking for increased mobility after phosphatase treatment. (c) Determine if the protein of interest can be phosphorylated by GSK-3 in vitro. Recombinant or immunoprecipitated GSK-3 is incubated with immunoprecipitated protein and the protein is examined for phosphorylation.