Studying MAP Kinase pathways during early development of Xenopus laevis.
The following chapter describes several methods involved in the detection of MAPK activities and phosphorylated proteins during early development of Xenopus laevis. The Xenopus embryo provides a powerful platform for biochemical studies. We describe here basic methods of embryo manipulations such as egg fertilization, embryo growth and maintenance, microinjection of capped RNA and antisense morpholino oligonucleotides (AMOs), and isolation of explants. In addition, we describe methods to detect phosphorylated proteins, to analyze kinase activity, and to interfere with signaling pathways. Immunohistochemical staining performed on whole embryos or on tissue sections is an additional method for the detection of phosphorylated proteins in the developing embryo. Approaches to activate or inhibit MAPK activities including the ectopic expression of mutated isoforms of MAPK kinase, or the incubation of embryo explants with pharmacological inhibitors are described. Finally, we describe an in vitro kinase assay specifically designed for the Xenopus embryo.
Keren
Aviad
A
Department of Biochemistry, Faculty of Medicine, Rappaport Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa, Israel.
Bengal
Eyal
E
D000483:Alleles; D000818:Animals; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D005306:Fertilization; D055785:Gene Knockdown Techniques; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D048669:MAP Kinase Kinase 6 / Q000235:genetics; D020935:MAP Kinase Signaling System; D008866:Microsurgery; D016376:Oligonucleotides, Antisense / Q000235:genetics; D010060:Ovulation; D016612:Paraffin Embedding; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D014982:Xenopus laevis / Q000196:embryology* / Q000378:metabolism* / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
D017362:Cyclic AMP Response Element-Binding Protein; D016376:Oligonucleotides, Antisense; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D048051:p38 Mitogen-Activated Protein Kinases; D048669:MAP Kinase Kinase 6