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Sci Rep. 2015 Aug 3;5:12767. doi: 10.1038/srep12767.

Detection and Differentiation of Threonine- and Tyrosine-Monophosphorylated Forms of ERK1/2 by Capillary Isoelectric Focusing-Immunoassay.

Author information

  • 11] LVR-Hospital Essen, Department of Psychiatry and Psychotherapy, Faculty of Medicine, University of Duisburg-Essen, Essen, Germany [2] German Center for Neurodegenerative Diseases (DZNE), Research Site Goettingen, Germany [3] Dept. of Psychiatry and Psychotherapy, University Medical Center Goettingen (UMG), Georg-August-University Goettingen, Germany.
  • 2LVR-Hospital Essen, Department of Psychiatry and Psychotherapy, Faculty of Medicine, University of Duisburg-Essen, Essen, Germany.
  • 3Department of Neurology, University of Ulm, Germany.
  • 41] Dept. of Psychiatry and Psychotherapy, University Medical Center Goettingen (UMG), Georg-August-University Goettingen, Germany [2] German Center for Neurodegenerative Diseases (DZNE), Research Site Goettingen, Germany.
  • 51] LVR-Hospital Essen, Department of Psychiatry and Psychotherapy, Faculty of Medicine, University of Duisburg-Essen, Essen, Germany [2] Dept. of Psychiatry and Psychotherapy, University Medical Center Goettingen (UMG), Georg-August-University Goettingen, Germany.

Abstract

The extracellular signal regulated kinases ERK1/2 play important roles in the regulation of diverse cellular functions and have been implicated in several human diseases. In addition to the fully activated, diphosphorylated ERK1/2 protein, monophosphorylated forms of ERK1/2 have been observed, which may have distinct biological functions. We report here on the highly sensitive detection and differentiation of unphosphorylated, threonine-phosphorylated (pT), tyrosine-phosphorylated (pY) and diphosphorylated ERK1 and ERK2 by capillary isoelectric focusing followed by immunological detection (CIEF-immunoassay). Eight different phosphorylated and unphosphorylated forms of ERK1/2 were resolved according to charge. The unequivocal identification and differentiation of ERK1 and ERK2 forms monophosphorylated at either threonine or tyrosine was achieved by competitive blocking with specific phospho-peptides and different phosphorylation-sensitive antibodies. The suitability of the additional pT-ERK1/2 and pY-ERK1/2 differentiation for the time-resolved in-depth study of phospho-form distribution in response to specific stimuli is demonstrated in human neuroblastoma SH-SY5Y and monocytic THP-1 cell lines, and in human peripheral blood mononuclear cells.

PMID:
26235103
PMCID:
PMC4522687
DOI:
10.1038/srep12767
[PubMed - in process]
Free PMC Article
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