Cell Signaling Technology

Product Pathways - Lymphocyte Signaling

T Cell Signaling Antibody Sampler Kit #9382

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Zap-70 (D1C10E) XP® Rabbit mAb #3165 40 µl W IP IF-F F H M 70 Rabbit IgG
Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb #2717 40 µl W H M (R) (Hm) (Mk) (C) (B) (Dg) (Pg) (Hr) 70, 72 kDa Rabbit IgG
SLP-76 Antibody #4958 40 µl W IP IHC-P H M 76 Rabbit
Phospho-LAT (Tyr191) Antibody #3584 40 µl W IP H 36, 38 Rabbit
LAT Antibody #9166 40 µl W IP IHC-P F H M (R) 36, 38 Rabbit
Phospho-Lck (Tyr505) Antibody #2751 40 µl W IP H M 56 Rabbit
Lck (73A5) Rabbit mAb #2787 40 µl W IF-IC F H 56 KDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-F=Immunofluorescence (Frozen)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  C=Chicken  B=Bovine  Dg=Dog  Pg=Pig  Hr=Horse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Each antibody in the T Cell Signaling Antibody Sampler Kit recognizes only its specific target and does not cross-react with other family members with the following exceptions: Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb which also recognizes Syk when phosphorylated at Tyr352; and Phospho-Lck (Tyr505) Antibody which may cross-react with certain phosphorylated Src family members.

Western Blotting

Western Blotting

Western blot analysis of extracts from Jurkat cells treated with with either H2O2 (2mM for 2 minutes) or with λ phosphatase; and extracts from Ramos cells treated with anti-human IgM (12 μg/ml for 2 minutes) using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) Rabbit mAb #2717.

Western Blotting

Western Blotting

Western blot analysis of extracts from Jurkat cells (serum starved) treated with calf intestinal alkaline phosphatase (CIP) or H2O2 (2 mM), using Phospho-Lck (Tyr505) Antibody #2751 (upper) or total Lck Antibody #2752 (lower).

Western Blotting

Western Blotting

Western blot analysis of Jurkat cell lysates, using Lck (73A5) Rabbit mAb #2787.


Western Blotting

Western Blotting

Western blot analysis of extracts from Jurkat cells using Zap-70 (D1C10E) XP® Rabbit mAb #3165.

Western Blotting

Western Blotting

Western blot analysis of extracts from serum starved Jurkat cells, either untreated or anti-CD3 treated (10 µg/ml for 2 minutes), using Phospho-LAT (Tyr191) Antibody #3584.

Western Blotting

Western Blotting

Western blot analysis of extracts from Jurkat and BW5147cells, using SLP-76 Antibody #4958.


Western Blotting

Western Blotting

Western blot analysis of extracts from Jurkat cells, either untreated or treated with λ phosphatase, using LAT Antibody #9166.

Description

The T Cell Signaling Antibody Sampler Kit contains primary and secondary antibodies to perform four Western blots with each antibody.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding the amino terminus of human Zap-70; Tyr319 of human Zap-70; and Tyr505 of human Lck.Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence of human SLP-76; residues surrounding Tyr191 of human LAT; the central region of human LAT; and residues surrounding Tyr505 of human Lck. Antibodies are purified by protein A and peptide affinity chromatography.

Background

The Syk family protein tyrosine kinase Zap-70 is expressed in T and NK cells and 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 through autophosphorylation and transphosphorylation by the Src family tyrosine kinase Lck (2-6). Tyrosine phosphorylation correlates with increased Zap-70 kinase activity and downstream signaling events. Expression of Zap-70 is correlated with disease progression and survival in patients with chronic lymphocytic leukemia (7,8).

LAT, a transmembrane adaptor protein expressed in T, NK and mast cells, is an important mediator for T cell receptor (TCR) signaling (9). Upon TCR engagement, activated Zap-70 phosphorylates LAT at multiple conserved tyrosine residues within SH2 binding motifs, exposing these motifs as the docking sites for downstream signaling targets (10,11). The phosphorylation of LAT at Tyr171 and 191 enables the binding of Grb2, Gads/SLP-76, PLCgamma1 and PI3 kinase through their SH2 domain and translocates them to the membrane. This process eventually leads to activation of the corresponding signaling pathways (11-12).

  1. Chu, D.H. et al. (1998) Immunol. Rev. 165, 167-180.
  2. Iwashima, M. et al. (1994) Science 263, 1136-1139.
  3. Neumeister, E.N. et al. (1995) Mol. Cell Biol. 15, 3171-3178.
  4. Chan, A.C. et al. (1995) EMBO J. 14, 2499-2508.
  5. Williams, B.L. et al. (1999) EMBO J. 18, 1832-1844.
  6. Di Bartolo, V. et al. (1999) J. Biol. Chem. 274, 6285-6294.
  7. Wiestner, A. et al. (2003) Blood 101, 4944-4951.
  8. Crespo, M. et al. (2003) N. Engl. J. Med. 348, 1764-1775.
  9. Wonerow, P. and Watson, S.P. (2001) Oncogene 20, 6273-83.
  10. Zhang, W. et al. (1998) Cell 92, 83-92.
  11. Paz, P.E. et al. (2001) Biochem J 356, 461-71.
  12. Zhang, W. et al. (2000) J Biol Chem 275, 23355-61.

Application References

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