Phospho-Stat4 (Tyr693) (D2E4)

4134:

ChIP Agarose , ChIP Magnetic , Flow , Immunofluorescence* , Immunoprecipitation , Western Blotting

* Product-specific protocol.

Phospho-Stat4 (Tyr693) (D2E4) Rabbit mAb recognizes endogenous levels of Stat4 protein only when phosphorylated at Tyr693.

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr693 of human Stat4 protein.

Western Blotting

Western blot analysis of extracts from NK-92 cells, untreated (-) or treated (+) with Human Interleukin-2 (hIL-2) #8907 (10 ng/ml, 15 min) or human interleukin-12 (hIL-12, 50 ng/ml, 15 min), using Phospho-Stat4 (Tyr693) (D2E4) Rabbit mAb (upper) and Stat4 (C46B10) Rabbit mAb #2653 (lower).

Flow Cytometry

Flow cytometric analysis of NK-92 cells, untreated (blue) or treated with IL-12 (green), using Phospho-Stat4 (Tyr693) (D2E4) Rabbit mAb.

IF-IC

Confocal immunofluorescent analysis of NK-92 cells, starved of IL-2 (5 hr) and then either untreated (upper) or IL-12-treated (50 ng/mL, 15 min; lower), using Phospho-Stat4 (Tyr693) (D2E4) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red).

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 10⁶ NK-92 cells starved of IL-2 overnight then treated with IL-12 (10 ng/ml, 4 hr) and either 20 μl of Phospho-Stat4 (Tyr693) (D2E4) Rabbit mAb or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP^® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human IRF-1 promoter primers, SimpleChIP^® Human PRF1 Promoter Primers #9014, and SimpleChIP^® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

The Jak-Stat signaling pathway is utilized by a large number of cytokines, growth factors, and hormones (1). Receptor-mediated tyrosine phosphorylation of Jak family members triggers phosphorylation of Stat proteins, resulting in their nuclear translocation, binding to specific DNA elements, and subsequent activation of transcription. The remarkable range and specificity of responses regulated by the Stats is determined, in part, by the tissue-specific expression of different cytokine receptors, Jaks, and Stats, as well as by the combinatorial coupling of various Stat members to different receptors (2). Stat4 is predominantly expressed in the spleen, thymus, and testis and has been most extensively investigated as the mediator of IL-12 responses (3-8). Activation of Stat4 is associated with phosphorylation at Tyr693 (9).

Stat4 is activated in response to IL-2 in natural killer (NK) cells, but not in T cells (10).

1. Darnell, J.E. et al. (1994) Science 264, 1415-1421.
2. Leonard, W.J. and O'Shea, J.J. (1998) Annu. Rev. Immunol. 16, 293-322.
3. Zhong, Z. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 4806-4810.
4. Yamamoto, K. et al. (1994) Mol. Cell Biol. 14, 4342-4349.
5. Jacobson, N.G. et al. (1995) J. Exp. Med. 181, 1755-1762.
6. Bacon, C.M. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7307-7311.
7. Thierfelder, W.E. et al. (1996) Nature 382, 171-174.
8. Kaplan, M.H. et al. (1996) Nature 382, 174-177.
9. Visconti, R. et al. (2000) Blood 96, 1844-52.
10. Wang, K.S. et al. (1999) J Immunol 162, 299-304.

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• 5267 Phospho-Stat4 (Tyr693) Antibody
• 2653 Stat4 (C46B10) Rabbit mAb
• 5097 Stat4 (2A2) Mouse mAb
• 7071 Phototope^® -HRP Western Blot Detection System, Anti-rabbit IgG, HRP-linked Antibody
• 7074 Anti-rabbit IgG, HRP-linked Antibody
• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 7003 20X LumiGLO^® Reagent and 20X Peroxide

For Research Use Only. Not For Use In Diagnostic Procedures.