Revision 1

#67748Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

20

SOURCE:

Rabbit

UniProt ID:

#P20963

Entrez-Gene Id:

919

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

Phospho-CD3ζ (Tyr142) Antibody recognizes endogenous levels of CD3ζ protein only when phosphorylated at Tyr142. This antibody cross-reacts with a 65 kDa phosphoprotein of unknown identity.

Species Reactivity:

Human

Species predicted to react based on 100% sequence homology

Pig

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr142 of human CD3ζ protein. Antibodies are purified by peptide affinity chromatography.

Background

When T cells encounter antigens via the T cell receptor (TCR), information about the quantity and quality of antigens is relayed to the intracellular signal transduction machinery (1). This activation process depends mainly on CD3 (Cluster of Differentiation 3), a multiunit protein complex that directly associates with the TCR. CD3 is composed of four polypeptides: ζ, γ, ε, and δ. Each of these polypeptides contains at least one immunoreceptor tyrosine-based activation motif (ITAM) (2). Engagement of the TCR complex with foreign antigens induces tyrosine phosphorylation in the ITAM motifs and phosphorylated ITAMs function as docking sites for signaling molecules such as ZAP-70 and the p85 subunit of PI-3 kinase (3,4). TCR ligation also induces a conformational change in CD3ε, such that a proline region is exposed and then associates with the adaptor protein Nck (5).

The CD3ζ invariant chain is a type-I transmembrane protein that exists in the TCR signaling complex as a disulfide-linked homodimer (6). The cytoplasmic tail of each CD3ζ monomer contains three distinct ITAM motifs, each containing two tyrosine residues. Phosphorylation of CD3ζ ITAM tyrosine residues, including Y142, is driven by recruitment of the Lck and Fyn tyrosine kinases to the TCR (7). Lck/Fyn-mediated ITAM phosphorylation creates docking sites that promote the SH2 domain-dependent recruitment and activation of Zap-70 (8-10), which drives amplification of signaling events downstream of the TCR that facilitate T cell activation (10). Phosphorylation of a pool of p16 CD3ζ leads to the generation of p21 and p23 species, which differ in the degree of ITAM phosphorylation. It has been proposed that the ratio of p21/p23 contributes to regulating the amplitude of T cell activation (11). CD3ζ plays an important role in the assembly and surface expression of the TCR complex. Indeed, research studies have demonstrated that CD3ζ is degraded in response to Ag-dependent TCR stimulation as a mechanism to tightly control T cell activation (12).

  1. Kuhns, M.S. et al. (2006) Immunity 24, 133-139.
  2. Pitcher, L.A. and van Oers, N.S. (2003) Trends Immunol. 24, 554-560.
  3. Osman, N. et al. (1996) Eur. J. Immunol. 26, 1063-1068.
  4. Hatada, M.H. et al. (1995) Nature 377, 32-38.
  5. Gil, D. et al. (2002) Cell 109, 901-912.
  6. Call, M.E. et al. (2006) Cell 127, 355-68.
  7. Housden, H.R. et al. (2003) Eur J Biochem 270, 2369-76.
  8. Hatada, M.H. et al. (1995) Nature 377, 32-8.
  9. Visco, C. et al. (2000) Biochemistry 39, 2784-91.
  10. Iwashima, M. et al. (1994) Science 263, 1136-9.
  11. Pitcher, L.A. et al. (2003) Immunol Rev 191, 47-61.
  12. Dumont, C. et al. (2002) J Immunol 169, 1705-12.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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Revision 1
#67748

Phospho-CD3ζ (Tyr142) Antibody

Western Blotting Image 1: Phospho-CD3ζ (Tyr142) Antibody Expand Image
Western blot analysis of extracts from serum-starved Jurkat cells, untreated (-) or treated with either H2O2 (11 mM, 1 min; +) or with cross-linked anti-CD3 plus anti-CD28 (10 μg/mL each, 5 min; +), using Phospho-CD3ζ (Tyr142) Antibody (upper) and CD3ζ Antibody (lower).
Western Blotting Image 2: Phospho-CD3ζ (Tyr142) Antibody Expand Image
Western blot analysis of extracts from serum-starved Jurkat cells, treated with either isotype control (-) or with cross-linked anti-CD3 plus anti-CD28 (10 ug/mL each, 5 min; +), using Phospho-CD3ζ (Tyr142) Antibody. The phospho-specificity of the antibody was verified by pre-incubating the antibody with no peptide (left), CD3ζ (Tyr142) non-phosphopeptide (middle), and CD3ζ (Tyr142) phosphopeptide (right).
Western Blotting Image 3: Phospho-CD3ζ (Tyr142) Antibody Expand Image
Western blot analysis of extracts from serum-starved Jurkat cells, untreated (-) or treated with H2O2 (11 mM, 1 min; +), using Phospho-CD3ζ (Tyr142) Antibody (upper) and CD3ζ Antibody (lower). H2O2-treated lysates were treated with calf intestinal phosphatase and Lambda phosphatase (CIP/λ phosphatase; +) as indicated.