Analysis of novel phospho-ITAM specific antibodies in a S2 reconstitution system for TCR-CD3 signalling.
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 zeta. Expressing TCR-CD3 subunits and tyrosine mutants thereof we tested the specificity of the novel antibodies in Western blot and immunopurification experiments. In particular, we generated and characterized the monoclonal antibody EM-26 that specifically recognizes phosphorylation of the membrane proximal tyrosine of zeta (phospho-zetaY1) and antisera raised against the first and the second phospho-tyrosine of CD3epsilon (phospho-epsilonY1 and phospho-epsilonY2).
Dopfer
Elaine P
EP
Department for Molecular Immunology, Max Planck-Institute for Immunobiology, Freiburg, Germany.
Schöpf
Barbara
B
Louis-Dit-Sully
Christine
C
Dengler
Eva
E
Höhne
Kerstin
K
Klescová
Andrea
A
Prouza
Marek
M
Suchanek
Miloslav
M
Reth
Michael
M
Schamel
Wolfgang W A
WW
D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000276:immunology*; D002460:Cell Line; D004330:Drosophila; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D015151:Immunoblotting; D051379:Mice; D010766:Phosphorylation; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000276:immunology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology*
D045163:Antibodies, Phospho-Specific; D017260:Receptor-CD3 Complex, Antigen, T-Cell