By Malcolm Whitman, Amy Sater
Developmental biologists were pushed to enquire progress issue signaling in embryos with the intention to comprehend the regulatory mechanisms underlying a given developmental procedure. hence, it truly is serious to discover the technical equipment and experimental designs for progress issue signaling in embryos. concentrating on particular pathways or pathway elements, research of development issue Signaling in Embryos presents the equipment and instructions for experimental layout to review significant facets of telephone signaling in vertebrate embryos. The e-book covers a vast variety of themes in signaling and numerous present version organisms. part I explores particular signaling pathways or pathway elements. during this part, a few chapters spotlight the biochemistry of signaling pathways in the course of improvement, that is frequently distinct from that saw in phone tradition platforms. part II discusses ionic regulatory mechanisms and the 2 chapters in part III learn methods of investigating gene rules based on extracellular indications. eventually, part IV addresses rising thoughts that facilitate built-in analyses of phone signaling in vivo in embryonic structures. that includes contributions from specialist researchers, research of progress issue Signaling in Embryos will supply a origin for additional explorations of the mobile regulatory mechanisms governing vertebrate embryonic improvement.
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Additional info for Analysis of Growth Factor Signaling in Embryos (Methods in Signal Transduction Series)
Axin, a negative regulator of the Wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of β-catenin, J Biol Chem 273 (18), 10823–6, 1998. 46. , Axin, an inhibitor of the Wnt signalling pathway, interacts with βcatenin, GSK-3β and APC and reduces the β-catenin level, Genes Cells 3 (6), 395–403, 1998. 47. , Clements, W. , Crystal structure of a βcatenin/Axin complex suggests a mechanism for the β-catenin destruction complex, Genes Dev 17 (22), 2753–64, 2003.
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Embryos are injected at the one- to two-cell stage with mRNA encoding experimental proteins or epitope-tagged β-catenin (step 1). After 4 hours, ectopic (tagged) or endogenous β-catenin is immunoprecipitated (step 2) and its phosphorylation state is examined by Western blotting using antibodies specific for phosphorylated β-catenin (step 3; see text). (c) Testing GSK-3 kinase activity indirectly by examination of β-catenin stability. Injected embryos (step 1) are lysed and either the membrane-bound β-catenin is removed by incubating the cytosolic fraction with ConA beads, or the cytosolic fraction is immunoprecipitated directly by incubating with GST-cadherin or GST-Tcf (step 2).