Product Pathways - Lymphocyte Signaling
Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody #2701
| Applications | Reactivity | MW (kDa) | Source |
|---|---|---|---|
| W IP IF-IC F | H M | 70 Zap-70, 72 Syk | Rabbit |
Applications Key:
W=Western Blotting
IP=Immunoprecipitation
IF-IC=Immunofluorescence (Immunocytochemistry)
F=Flow Cytometry
Reactivity Key:
H=Human
M=Mouse
Species enclosed in parentheses are predicted to react based on 100% sequence homology. Species cross-reactivity is determined by Western blot.
Specificity / Sensitivity
Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody detects endogenous levels of Zap-70 only when phosphorylated at Tyr319. It cross-reacts with endogenous levels of Syk when phosphorylated at Tyr352.
Source / Purification
Polyclonal antibodies are produced by immunizing rabbits with a synthetic phospho-peptide (KLH-coupled) corresponding to residues surrounding Tyr319 of human Zap-70. Antibodies are purified by protein A and peptide affinity chromatography.
Western Blotting
Western blot analysis of extracts from Jurkat cells, starved for 16 hours, and treated with 2 mM H2O2 or with calf intestinal alkaline phosphatase (CIP), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody (upper) or control Zap-70 Antibody #2702 (lower).
Western Blotting
Western blot analysis of extracts from Ramos cells, untreated or treated with anti-IgM (12 µg/ ml for 2 minutes), hydrogen peroxide (10 mM for 2 minutes) or lambda phosphatase, using Phospho-Zap-70 (Tyr319)/ Syk (Tyr352) Antibody.
Flow Cytometry
Two-color flow cytometric analysis of Jurkat cells, untreated (left) or anti-CD3 activated (right), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody and Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) mAb #9106. Anti-CD3 activation increases the intensity of label with both antibodies.
Flow Cytometry
Flow cytometric analysis of Jurkat cells, untreated (blue) or CD3 treated (green), using Phospho-Zap70 (Tyr319)/Syk (Tyr352) Antibody compared to a nonspecific negative control antibody (red).
IF-IC
Immunofluorescent analysis of Jurkat cells, CD3-treated (left) or untreated (right), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).
Background
Zap-70, a Syk family protein tyrosine kinase expressed in T and NK cells, plays a critical role in mediating T cell activation in response to T cell receptor (TCR) engagement (1). Following TCR engagement, Zap-70 is rapidly phosphorylated on several tyrosine residues, presumably by two mechanisms: autophosphorylation and transphosphorylation by the Src family tyrosine kinase, Lck (2-6). Tyrosine phosphorylation of Zap-70 correlates with its increased kinase activity and downstream signaling events. In patients with chronic lymphocytic leukemia (CLL), total Zap-70 expression was shown to be correlated with disease progression and survival (7,8).
Phosphorylation of Tyr319 is required for the assembly of a Zap-70-containing signaling complex that leads to the activation of the PLC-gamma1-dependent and Ras-dependent signaling cascades in antigen-stimulated T cells (5,6). The orthologous Tyr352 residue in Syk is also involved in the association with PLC-gamma1 (9).
- Chu, D.H. et al. (1998) Immunol. Rev. 165, 167-180.
- Iwashima, M. et al. (1994) Science 263, 1136-1139.
- Neumeister, E.N. et al. (1995) Mol. Cell Biol. 15, 3171-3178.
- Chan, A.C. et al. (1995) EMBO J. 14, 2499-2508.
- Williams, B.L. et al. (1999) EMBO J. 18, 1832-1844.
- Di Bartolo, V. et al. (1999) J. Biol. Chem. 274, 6285-6294.
- Wiestner, A. et al. (2003) Blood 101, 4944-4951.
- Crespo, M. et al. (2003) N. Engl. J. Med. 348, 1764-1775.
- Law, C. L. et al. (1996) Mol. Cell. Biol. 16, 1305-1315.
Application References
- Guntermann, C. and Alexander, D.R. (2002) CTLA-4 suppresses proximal TCR signaling in resting human CD4(+) T cells by inhibiting ZAP-70 Tyr(319) phosphorylation: A potential role for tyrosine phosphatases. J. Immunol. 168, 4420-4429. This article references the use of Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody in the following applications: IP Western Blotting
- Houtman, J. C. et al. (2005) Early phosphorylation kinetics of proteins involved in proximal TCR-mediated signaling pathways. J Immunol. 175 (4), 2449-2458. This article references the use of Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody in the following applications: Western Blotting
- Schade, A.E. and Levine, A.D. (2002) Lipid Raft Heterogeneity in Human Peripheral Blood T Lymphoblasts: A Mechanism for Regulating the Initiation of TCR Signal Transduction. J. Immunol. 168, 2233-2239. This article references the use of Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody in the following applications: Western Blotting
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