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Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe


Reference:

Sipthorp, J., Lebraud, H., Gilley, R., Kidger, A. M., Okkenhaug, H., Saba-El-Leil, M., Meloche, S., Caunt, C. J., Cook, S. J. and Heightman, T. D., 2017. Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe. Bioconjugate Chemistry, 28 (6), pp. 1677-1683.

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http://dx.doi.org/10.1021/acs.bioconjchem.7b00152

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Abstract

The RAS-RAF-MEK-ERK pathway has been intensively studied in oncology, with RAS known to be mutated in ∼30% of all human cancers. The recent emergence of ERK1/2 inhibitors and their ongoing clinical investigation demands a better understanding of ERK1/2 behavior following small-molecule inhibition. Although fluorescent fusion proteins and fluorescent antibodies are well-established methods of visualizing proteins, we show that ERK1/2 can be visualized via a less-invasive approach based on a two-step process using inverse electron demand Diels-Alder cycloaddition. Our previously reported trans-cyclooctene-tagged covalent ERK1/2 inhibitor was used in a series of imaging experiments following a click reaction with a tetrazine-tagged fluorescent dye. Although limitations were encountered with this approach, endogenous ERK1/2 was successfully imaged in cells, and "on-target" staining was confirmed by over-expressing DUSP5, a nuclear ERK1/2 phosphatase that anchors ERK1/2 in the nucleus.

Details

Item Type Articles
CreatorsSipthorp, J., Lebraud, H., Gilley, R., Kidger, A. M., Okkenhaug, H., Saba-El-Leil, M., Meloche, S., Caunt, C. J., Cook, S. J. and Heightman, T. D.
DOI10.1021/acs.bioconjchem.7b00152
Related URLs
URLURL Type
http://www.scopus.com/inward/record.url?scp=85021164682&partnerID=8YFLogxKUNSPECIFIED
Uncontrolled Keywordsbiotechnology,bioengineering,biomedical engineering,pharmacology,pharmaceutical science,organic chemistry
DepartmentsFaculty of Science > Biology & Biochemistry
RefereedYes
StatusPublished
ID Code56642

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