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Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment
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Assaying Activation and Subcellular Localization of ERK in Cells and Tissues

Carme Caelles1, Carles Bayod2, Melisa Morcillo2

  • 1Department of Biochemistry and Physiology, School of Pharmacy, University of Barcelona, Av. Joan XXIII, 27-31, 08028, Barcelona, Spain. ccaelles@ub.edu.

Methods in Molecular Biology (Clifton, N.J.)
|December 8, 2016
PubMed

Insights

Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are key in cell processes. This chapter details methods to assay ERK activity and monitor its activation through phosphorylation and nuclear translocation.

Area of Science:

  • Molecular Biology
  • Cell Signaling

Background:

  • Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are crucial in cell proliferation, differentiation, and oncogenic transformation.
  • ERK1/2 are part of the mitogen-activated protein kinase (MAPK) family, activated by dual phosphorylation.
  • ERK activation involves dimerization and nuclear translocation.

Purpose of the Study:

  • To describe established methods for assaying ERK activity.
  • To present techniques for monitoring ERK activation.
  • To detail a procedure for observing ERK nuclear translocation.

Main Methods:

  • Assaying ERK activity via immunocomplex phosphorylation of a substrate.
  • Immunoblot analysis using phospho-specific antibodies for the Thr-X-Tyr motif.
  • Immunocytochemistry to detect stimulus-induced nuclear translocation of ERK1/2.

Main Results:

  • Established protocols allow for the measurement of ERK kinase activity.
  • Specific antibodies enable the detection of activated ERK1/2.
  • Immunocytochemistry visualizes the dynamic nuclear import of ERK1/2.

Conclusions:

  • The described methods provide robust tools for studying ERK1/2 signaling pathways.
  • These techniques are valuable for research in physiological and pathological contexts.
  • Understanding ERK activation mechanisms is critical for therapeutic target identification.
Keywords:
ERK activityERK nuclear translocationERK1ERK2Immunoblot analysisImmunocomplex assayImmunocytochemistry analysisPhospho-ERK

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