Abstract
The mitogen-activated protein kinase, ERK is activated by a dual phosphorylation on threonine and tyrosine residues. Using a synthetic diphospho peptide, we have generated a monoclonal antibody directed to the active ERK. The antibody specifically identified the active doubly phosphorylated, but not the inactive mono- or non- phosphorylated forms of ERKs. A direct correlation was observed between ERK activity and the intensity in Western blot of mitogen-activated protein kinases from several species. The antibody was proven suitable for immunofluorescence staining, revealing a transient reactivity with ERKs that were translocated to the nucleus upon stimulation. In conclusion, the antibody can serve as a useful tool in the study of ERK signaling in a wide variety of organisms.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3T3 Cells
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Animals
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Antibodies, Monoclonal*
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Antibody Specificity
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Binding Sites, Antibody
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Blotting, Western
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COS Cells
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism
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Cell Line
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Drosophila melanogaster
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Enzyme Activation
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Epidermal Growth Factor / pharmacology
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Eukaryota
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HeLa Cells
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Humans
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Kinetics
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Mice
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Mitogen-Activated Protein Kinases / metabolism*
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Mutagenesis, Site-Directed
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Nerve Tissue Proteins / metabolism*
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Phosphorylation
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Point Mutation
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Rats
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae / enzymology
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Signal Transduction
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Spodoptera
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Threonine
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Transfection
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Tyrosine
Substances
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Antibodies, Monoclonal
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Nerve Tissue Proteins
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Recombinant Proteins
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Threonine
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Tyrosine
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Epidermal Growth Factor
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Calcium-Calmodulin-Dependent Protein Kinases
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Mitogen-Activated Protein Kinases