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57th Annual Meeting of the German Society for Neuropathology and Neuroanatomy (DGNN)

German Society for Neuropathology and Neuroanatomy

12. - 15.09.2012, Erlangen

57th Annual Meeting of the German Society for Neuropathology and Neuroanatomy (DGNN)

Capillary IEF for analyzing the MAP kinase phosphorylation patterns in an in vitro model for microglial inflammation response

Meeting Abstract

  • presenting/speaker Inga Kraus - LVR-Klinikum Essen, Labor für molekulare Neurobiologie, Essen, Germany
  • Just Genius - LVR-Klinikum Essen, Labor für molekulare Neurobiologie, Essen, Germany
  • Hans Klafki - LVR-Klinikum Essen, Labor für molekulare Neurobiologie, Essen, Germany
  • Jens Wiltfang - LVR-Klinikum Essen, Labor für molekulare Neurobiologie, Essen, Germany

Deutsche Gesellschaft für Neuropathologie und Neuroanatomie. 57th Annual Meeting of the German Society for Neuropathology and Neuroanatomy (DGNN). Erlangen, 12.-15.09.2012. Düsseldorf: German Medical Science GMS Publishing House; 2012. Doc12dgnnPP2.6

DOI: 10.3205/12dgnn042, URN: urn:nbn:de:0183-12dgnn0427

Published: September 11, 2012

© 2012 Kraus et al.
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Outline

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Background: The formation of amyloid plaques has been suggested to be associated with an inflammatory reaction. Microglial inflammation responses may be triggered by an interaction with these plaques, a mechanism which seems to be essential for the pathophysiology of Alzheimer's disease. The monocytic cell line THP-1 was employed as a model for microglial responses. As monocytes are immunologically active, the kinetics of signaling via phosphorylation or dephosphorylation of p38 MAP kinase is of vital interest for AD-related responses. Like ERK1/2 and JNK, p38 belongs to the family of the mitogen-activated protein kinases and is also known as one of the stress-related kinases. It responds to many inflammatory stimuli, e.g. IL-1ß, TNF-α, IFN-γ, etc. The p38 MAPK responses were functionally characterized in cellular assays as well as on the level of protein biochemistry.

Methods: To investigate the alteration of the phosphorylation state of p38 MAPK, THP-1 cells were first treated with anisomycin and a range of physiological activators for different incubation times. Subsequently using the capillary-based, robotic Nanoimmunoassay the phosphoproteins were separated according to their isoelectric point and then immunostained with a choice of antibodies directed against the phosphorylated and unphosphorylated forms of p38 MAPK. For fluorescence microscopy the cells were treated with anisomycin, immobilized on the slides and immunostained with an anti-phospho-p38-Ab. Western blot analysis were used to validate the phosphorylation.

Results: Western blot analysis revealed a marked increase in p38 MAPK phosphorylation after treatment with anisomycin. Using the Nanoimmunoassay two significant phosphorylation peaks were detected which could be analyzed in a semiquantitative manner. Furthermore, the fluorescence microscopy may reflect an alteration of the p38 MAPK localization within the cells.

Conclusions: The methods chosen proved to be applicable for the assessment of p38 MAPK phosphorylation. The two signals obtained within the Nanoimmunoassay probably represent the mono- and diphosphorylated forms of the p38 MAP kinase. The rearrangement of the fluorescence signal may display the dimerization of phospho-p38 MAPK and its translocation from the cytoplasm to the nucleus.