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Your search keyword '"Antibodies, Phospho-Specific"' showing total 4 results
Start Over Descriptor "Antibodies, Phospho-Specific" Publisher elsevier
4 results on '"Antibodies, Phospho-Specific"'

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

Author

Elaine P. Dopfer, Barbara Schöpf, Andrea Kleščová, Marek Prouza, Eva Dengler, Wolfgang W. A. Schamel, Michael Reth, Kerstin Höhne, Christine Louis-Dit-Sully, and Miloslav Suchánek

Subjects
medicine.drug_class, T-Lymphocytes, Immunology, Genetic Vectors, Immunoblotting, Enzyme-Linked Immunosorbent Assay, Biology, Monoclonal antibody, Receptor tyrosine kinase, Cell Line, 03 medical and health sciences, Mice, 0302 clinical medicine, Antigen, Immunoreceptor tyrosine-based activation motif, medicine, Immunology and Allergy, Animals, Tyrosine, Phosphorylation, 030304 developmental biology, 0303 health sciences, Kinase, 3. Good health, Biochemistry, Gene Expression Regulation, Receptor-CD3 Complex, Antigen, T-Cell, biology.protein, Drosophila, Signal transduction, Antibodies, Phospho-Specific, 030215 immunology, Signal Transduction
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 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).

Published
2010
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2. The 4G10, pY20 and p-TYR-100 antibody specificity: profiling by peptide microarrays

Author

Tinti, M, Castagnoli, L, Nardozza, A, Ferrari, E, Cesareni, G, Sacco, F, and Corallino, S

Subjects
Proteomics, Phosphopeptides, Cell, Molecular Sequence Data, Protein Array Analysis, Bioengineering, Peptide, Antibodies, Phospho-Specific, Antibody Specificity, Signal Processing, Computer-Assisted, Reproducibility of Results, Phosphorylation, Humans, HEK293 Cells, Amino Acid Sequence, Biology, Antibodies, 03 medical and health sciences, 0302 clinical medicine, Computer-Assisted, medicine, Tyrosine, Molecular Biology, Peptide sequence, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Phospho-Specific, HEK 293 cells, General Medicine, 3. Good health, Settore BIO/18 - Genetica, medicine.anatomical_structure, Biochemistry, chemistry, 030220 oncology & carcinogenesis, Signal Processing, Peptide microarray, DNA microarray, Biotechnology
Abstract

The reversible phosphorylation of tyrosine residues is one of the most frequent post-translational modifications regulating enzymatic activities and protein-protein interactions in eukaryotic cells. Cells responding to internal or external regulatory inputs modify their phosphorylation status and diseased cells can often be diagnosed by observing alterations in their qualitative or quantitative phosphorylation profile. As a consequence the ability to describe the phosphorylation profile of a cell is central to many approaches aiming at the characterisation of signalling pathways. Anti-phosphotyrosine (pY) antibodies are widely used as experimental tools to monitor the phosphorylation status of a cell. By using peptide microarray technology we have characterised the substrate specificity of three widely used pY antibodies. We report that they are more sensitive to sequence context than is generally assumed and that their sequence preferences differ.

Published
2012

3. The Balpha and Bdelta regulatory subunits of PP2A are necessary for assembly of the CaMKIV.PP2A signaling complex.

Author

Reece KM, Mazalouskas MD, and Wadzinski BE

Subjects
Antibodies, Phospho-Specific, Calcium-Calmodulin-Dependent Protein Kinase Type 4 genetics, Cell Line, Holoenzymes genetics, Holoenzymes metabolism, Humans, Mutation, Phosphorylation, Protein Phosphatase 2 genetics, Protein Subunits genetics, Signal Transduction, Calcium-Calmodulin-Dependent Protein Kinase Type 4 metabolism, Protein Phosphatase 2 metabolism, Protein Subunits metabolism
Abstract

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine kinase that is important in synaptic plasticity and T cell maturation. Activation of CaMKIV requires calcium/calmodulin binding and phosphorylation at T200 by CaMK kinase. Our previous work has shown that protein serine/threonine phosphatase 2A (PP2A) forms a complex with CaMKIV and negatively regulates its activity. Here we demonstrate that PP2A tightly regulates T200 phosphorylation of endogenous CaMKIV, but has little effect on the phosphorylation of the ectopically-expressed kinase. This differential regulation of endogenous versus exogenous CaMKIV is due to differences in their ability to associate with PP2A, as exogenous CaMKIV associates poorly with PP2A in comparison to endogenous CaMKIV. The inability of exogenous CaMKIV to associate with PP2A appears to be due to limiting amounts of endogenous PP2A regulatory B subunits, since coexpression of Balpha or Bdelta causes the recruitment of PP2Ac to ectopic CaMKIV, leading to formation of a CaMKIV.PP2A complex. Together, these data indicate that the B subunits are essential for the interaction of PP2A with CaMKIV.

Published
2009
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4. Phosphorylation state-specific antibodies: applications in investigative and diagnostic pathology.

Author

Mandell JW

Subjects
Animals, Antibody Specificity, Humans, Immunohistochemistry, Phosphorylation, Antibodies, Phospho-Specific, Neoplasms diagnosis, Pathology, Clinical methods, Proteins metabolism
Abstract

Until recently, the investigation of protein phosphorylation was limited to biochemical studies of enzyme activities in homogenized tissues. The availability of hundreds of phosphorylation state-specific antibodies (PSSAs) now makes possible the study of protein phosphorylation in situ, and is opening many exciting opportunities in investigative and diagnostic pathology. This review illustrates the power of PSSAs, especially in immunohistochemical applications to human disease and animal models. Technical considerations, including antibody specificity and lability of phosphoepitopes, are covered, along with potential pitfalls, illustrated by a case study. In the arena of oncology, PSSAs may prove especially valuable in directly demonstrating the efficacy of chemotherapies targeted at protein kinase cascades. Novel applications of PSSAs are also beginning to reveal molecular mechanisms of inflammatory, degenerative, and toxin-induced diseases.

Published
2003
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