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  Analysis of novel phospho-ITAM specific antibodies in a S2 reconstitution system for TCR-CD3 signalling

Dopfer, E. P., Schöpf, B., Louis-Dit-Sully, C., Dengler, E., Höhne, K., Klescová, A., et al. (2010). Analysis of novel phospho-ITAM specific antibodies in a S2 reconstitution system for TCR-CD3 signalling. Immunology Letters, 130, 43-50.

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 Creators:
Dopfer, Elaine P.1, Author           
Schöpf, Barbara1, Author           
Louis-Dit-Sully, Christine1, Author           
Dengler, Eva1, Author           
Höhne, Kerstin1, Author           
Klescová, Andrea, Author
Prouza, Marek, Author
Suchanek, Miloslav, Author
Reth, Michael1, Author           
Schamel, Wolfgang W. A.1, Author           
Affiliations:
1Research Group and Chair of Molecular Immunology of the University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society, ou_2243645              

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 Abstract: The T cell antigen receptor (TCR-CD3) complex contains 12 different cytoplasmic tyrosines, each of which is part of an immunoreceptor tyrosine-based activation motif and thus occurs in similar sequence context. Since phosphorylation of individual tyrosines can be correlated with the quality of the T cell response, monitoring their phosphorylation is important. We thus generated novel antibodies against phospho-tyrosines of the TCR-CD3 complex and tested the specificity in a synthetic biology approach. We utilized the Drosophila S2 reconstitution system testing several kinases and stimulation conditions that lead to optimal phosphorylation of the TCR-CD3 subunit ζ. Expressing TCR-CD3 subunits and tyrosine mutants thereof we tested the specificity of the novel antibodies in Western blot and immuno-purification experiments. In particular, we generated and characterized the monoclonal antibody EM-26 that specifically recognizes phosphorylation of the membrane proximal tyrosine of ζ (phospho-ζY1) and antisera raised against the first and the second phospho-tyrosine of CD3ε (phospho-εY1 and phospho-εY2).

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Language(s): eng - English
 Dates: 2010
 Publication Status: Issued
 Pages: -
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 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 527613
 Degree: -

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Title: Immunology Letters
  Alternative Title : Immunol. Lett.
Source Genre: Journal
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Pages: - Volume / Issue: 130 Sequence Number: - Start / End Page: 43 - 50 Identifier: -