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Full Description
This volume provides a collection of techniques and approaches for the study of ERK signaling. It begins with a historical perspective of genetic and molecular discoveries, followed by chapters covering specific topics in a broad range of experimental systems, including in vitro assays of EGFR and ERK activities; proteomic and genome-wide analyses of ERK signaling targets; cell biological, genetic, quantitative and imaging approaches in cells and model organisms; and integrated mathematical modeling of the ERK pathway. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Cutting-edge and thorough, ERK Signaling: Methods and Protocols will aid and stimulate further advances in the vibrant field of ERK signaling.
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Contents
How Genetics has Helped Piece Together the MAPK Signaling Pathway.- In Vitro Enzyme Kinetics Analysis of EGFR.- High-Throughput Analysis of Mammalian Receptor Tyrosine Kinase Activation in Yeast Cells.- Structural Studies of ERK2 Protein Complexes.- Isolation and Characterization of Intrinsically Active (MEK-Independent) Mutants of Mpk1/Erk.- Assaying Activation and Subcellular Localization of ERK in Cells and Tissues.- Detection and Functional Analysis of SUMO-Modified MEK.- Single-Step Affinity Purification of ERK Signaling Complexes Using the Streptavidin-Binding Peptide (SBP) Tag.- High-Throughput In Vitro Identification of Direct MAPK/Erk Substrates.- Global Identification of ERK Substrates by Phosphoproteomics Based on IMAC and 2D-DIGE.- Analysis of Ras/ERK Compartmentalization by Subcellular Fractionation.- Cell-Based Assays to Study ERK Pathway/Caveolin1 Interactions.- The Nuclear Translocation of ERK.- Visualization of RAS/MAPK Signaling In Situ by the Proximity Ligation Assay (PLA).- Measuring ERK Activity Dynamics in Single Living Cells Using FRET Biosensors.- Quantifying Tensile Force and ERK Phosphorylation on Actin Stress Fibers.- Co-Culture Activation of MAP Kinase in Drosophila S2 Cells.- Isolation of Mouse Embryonic Stem Cell Lines in the Study of ERK1/2 MAP Kinase Signaling.- 3D Organotypic Culture Model to Study Components of ERK Signaling.- Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.- Genome-Wide Analysis of RAS/ERK Signaling Targets.- Probing Chromatin Modifications in Response to ERK Signaling.- Analyzing pERK Activation during Planarian Regeneration.- Discovering Functional ERK Substrates Regulating Caenorhabditis elegans Germline Development.- Reconstructing ERK Signaling in the Drosophila Embryo from Fixed Images.- Using CRISPR-Cas9 to Study ERK Signaling in Drosophila.- Analyzing ERK Signal Dynamics during Zebrafish Somitogenesis.- Modeling RASopathies with Genetically Modified Mouse Models.- Dissecting Cell-Fate Determination through Integrated Mathematical Modeling of the ERK/MAPK Signaling Pathway.
