Assaying Activation and Subcellular Localization of ERK in Cells and Tissues

  • Carme CaellesEmail author
  • Carles Bayod
  • Melisa Morcillo
Part of the Methods in Molecular Biology book series (MIMB, volume 1487)


The extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are the focus of many studies due to their involvement in numerous physiological and pathological processes, such as cell proliferation and differentiation, and oncogenic transformation, respectively. ERK1/2 belong to the mitogen-activated protein kinase (MAPKs) family, which are serine/threonine kinases that participate in signal transduction and are activated by dual phosphorylation in the Thr-X-Tyr motif located in their activation loop. In addition, ERK activation induces its dimerization and translocation into the nucleus. On the basis of this knowledge, different assays and tools have been developed to determine ERK activity or monitor its activation. In this chapter, we describe methods to assay ERK activity based on the ability of ERK immunocomplexes to phosphorylate a substrate, as well as on immunoblot analysis using antibodies that recognize ERK1/2 phosphorylated in the Thr-X-Tyr motif. In addition, we describe an immunocytochemistry procedure to reveal stimuli-induced nuclear translocation of ERK1/2.

Key words

ERK1 ERK2 ERK activity Phospho-ERK ERK nuclear translocation Immunocomplex assay Immunoblot analysis Immunocytochemistry analysis 



This work was supported by the Plan Nacional I + D grants SAF2013-40832-P and SAF2014-57856-P from the Spanish Ministry of Economy and Competitivity. C.B. was supported by an ADR predoctoral fellowship from University of Barcelona, Spain.


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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Biochemistry and PhysiologySchool of Pharmacy, University of BarcelonaBarcelonaSpain

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