Phospho-Stat1 (Ser727) Antibod

9177:

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

* Product-specific protocol.

Phospho-Stat1 (Ser727) Antibody detects endogenous levels of Stat1α only when phosphorylated at Ser727. This site is deleted in Stat1β. This antibody does not significantly cross-react with the corresponding phosphorylated residues of other Stat proteins.

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser727 of human Stat1. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western blot analysis of extracts from 293 (human), NIH/3T3 (mouse), and C6 (rat) cells, untreated or UV-stimulated, using Phospho-Stat1 (Ser727) Antibody

Flow Cytometry

Flow cytometric analysis of Hela cells, phosphatase-treated (red), untreated (blue) or UV-treated (green), using Phospho-Stat1 (Ser727) Antibody.

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left), IFN-γ treated (center) or IFN-γ and phosphatase treated (right), labeled with Phospho-Stat1 (Ser727) Antibody (green) and Pan-Keratin (C11) Mouse mAb #4545 (red).

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 10⁶ HT-1080 cells treated with IFN-γ (50 ng/ml) for 30 minutes and either 20 μl of Phospho-Stat1 (Ser727) Antibody 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 TAP1 Promoter Primers #5148, 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 Stat1 transcription factor is activated in response to a large number of ligands (1) and is essential for responsiveness to IFN-α and IFN-γ (2,3). Phosphorylation of Stat1 at Tyr701 induces Stat1 dimerization, nuclear translocation, and DNA binding (4). Stat1 protein exists as a pair of isoforms, Stat1α (91 kDa) and the splice variant Stat1β (84 kDa). In most cells, both isoforms are activated by IFN-α, but only Stat1α is activated by IFN-γ. The inappropriate activation of Stat1 occurs in many tumors (5). In addition to tyrosine phosphorylation, Stat1 is also phosphorylated at Ser727 through a p38 mitogen-activated protein kinase (MAPK)-dependent pathway in response to IFN-α and other cellular stresses (6). Serine phosphorylation may be required for the maximal induction of Stat1-mediated gene activation.

1. Heim, M.H. (1999) J. Recept. Signal. Transduct. Res. 19, 75-120.
2. Durbin, J.E. et al. (1996) Cell 84, 443-450.
3. Meraz, M.A. et al. (1996) Cell 84, 431-442.
4. Ihle, J.N. et al. (1994) Trends Biochem. Sci. 19, 222-227.
5. Frank, D.A. (1999) Mol. Med. 5, 432-456.
6. Wen, Z. et al. (1995) Cell 82, 241-250.

• Bowie, M. L. et al. (2004) Interferon-regulatory factor-1 is critical for tamoxifen-mediated apoptosis in human mammary epithelial cells. Oncogene 23, 8743-8755. Applications: Western Blotting
• Kaizu, T. et al. (2008) Am J Physiol Gastrointest Liver Physiol 294, G236-44. Applications: Western Blotting

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