Cell Signaling Technology

Product Pathways - Jak/Stat Pathway

Phospho-Stat3 (Ser727) Antibody #9134

Applications Reactivity Sensitivity MW (kDa) Source
W IP IF-IC ChIP H M R (B) Endogenous 86 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  B=Bovine
Species cross-reactivity is determined by Western blot.

Protocols

Specificity / Sensitivity

Phospho-Stat3 (Ser727) Antibody detects endogenous levels of Stat3 only when phosphorylated at Ser727. The antibody does not significantly cross-react with the corresponding phospho-serines of other Stat proteins.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser727 of mouse Stat3. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from A431 cells, untreated or EGF-treated (100 ng/ml) for the indicated times, using Phospho-Stat3 (Ser727) Antibody (upper) or Stat3 Antibody #9132 (lower).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left), IL-6 treated (center) or IL-6 and phosphatase treated (right), labeled with Phospho-Stat3 (Ser727) Antibody (green) and Pan-Keratin (C11) Mouse mAb #4545 (red).

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HepG2 cells starved overnight and treated with IL-6 (100ng/ml) for 30 minutes, and either 20 μl of Phospho-Stat3 (Ser727) Antibody #9134 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 primers specific for the c-FOS gene and the heterochromatic α Satellite repeat element. 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.


Background

The Stat3 transcription factor is an important signaling molecule for many cytokines and growth-factor receptors (1) and is required for murine fetal development (2). Stat3 is constitutively activated in a number of human tumors (3,4) and possesses oncogenic potential (5) and anti-apoptotic activities (3). Stat3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation and DNA binding (6,7). Transcriptional activation seems to be regulated by phosphorylation at Ser727 through the MAPK or mTOR pathways (8,9). Stat3 isoform expression appears to reflect biological function as the relative expression levels of Stat3α (86 kDa) and Stat3β (79 kDa) depend on cell type, ligand exposure or cell maturation stage (10). It is notable that Stat3β lacks the serine phosphorylation site within the carboxy-terminal transcriptional activation domain (8).

  1. Heim, M.H. (1999) J. Recept. Signal Transduct. Res. 19, 75-120.
  2. Takeda, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 3801-3804.
  3. Catlett-Falcone, R. et al. (1999) Immunity 10, 105-115.
  4. Garcia, R. and Jove, R. (1998) J. Biomed. Sci. 5, 79-85.
  5. Bromberg, J.F. et al. (1999) Cell 98, 295-303.
  6. Darnell Jr., J.E. et al. (1994) Science 264, 1415-1421.
  7. Ihle, J.N. (1995) Nature 377, 591-594.
  8. Wen, Z. et al. (1995) Cell 82, 241-250.
  9. Yokogami, K. et al. (2000) Curr. Biol. 10, 47-50.
  10. Biethahn, S. et al. (1999) Exp. Hematol. 27, 885-894.

Application References

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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