Cell Signaling Technology

Product Pathways - Jak/Stat Pathway

Phospho-Stat3 (Tyr705) Antibody #9131

Applications Reactivity Sensitivity MW (kDa) Source
W IP IF-IC ChIP H M R Mk (C) (B) (Dg) Endogenous 79, 86 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  C=Chicken  B=Bovine  Dg=Dog
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

* Product-specific protocol.

Specificity / Sensitivity

Phospho-Stat3 (Tyr705) Antibody detects endogenous levels of Stat3 only when phosphorylated at Tyr705. The antibody does not cross-react with other Stat proteins when phosphorylated on the corresponding tyrosine residue, but has been shown to cross-react with Phospho-EGFR.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr705 of mouse Stat3. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from CNTF-treated SK-N-MC cells and EGF-treated A431 cells, using Phospho-Stat3 (Tyr705) Antibody (upper) or Stat3 Antibody #9132 (lower).

IP

IP

Immunoprecipitation of extracts from CNTF-treated 35S-labeled SK-N-MC cells, using Phospho-Stat3 (Tyr705) Antibody (upper) or Stat3 Antibody #9132 (lower).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells either serum-starved (left) or IFNalpha-treated (right), using Phospho-Stat3 (Tyr705) Antibody (green) and S6 Ribosomal Protein (54D2) Mouse mAb #2317 (red).


Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 Hep G2 cells starved overnight and treated with IL-6 (100 ng/ml) for 30 minutes, and either 10 μl of Phospho-Stat3 (Tyr705) 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 c-Fos Promoter Primers #4663, 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.

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. (2001) J Recept Signal Transduct Res 19, 75-120.
  2. Takeda, K. et al. (1997) Proc Natl Acad Sci U S A 94, 3801-4.
  3. Catlett-Falcone, R. et al. (1999) Immunity 10, 105-15.
  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, J.E. et al. (1994) Science 264, 1415-21.
  7. Ihle, J.N. (1995) Nature 377, 591-4.
  8. Wen, Z. et al. (1995) Cell 82, 241-50.
  9. Yokogami, K. et al. (2000) Curr Biol 10, 47-50.
  10. Biethahn, S. et al. (1999) Exp Hematol 27, 885-94.

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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