Cell Signaling Technology

Product Pathways - Jak/Stat Pathway

Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IHC-P IHC-F IF-IC F ChIP H M Endogenous 84, 91 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IHC-F=Immunohistochemistry (Frozen)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse
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-Stat1 (Tyr701) (58D6) Rabbit mAb detects endogenous levels of Stat1 only when phosphorylated at tyrosine 701. The antibody detects phosphorylated tyrosine 701 of p91 Stat1 and also the p84 splice variant. It does not cross-react with the corresponding phospho-tyrosines of other Stat proteins.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr701 of human Stat1.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells untreated or treated with interferon-α (IFN-α), using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb (upper) or Stat1 Antibody (#9172) (lower).

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded Non-Hodgkin's lymphoma control (left) or λ phosphatase treated (right), using Phospho-Stat1 (tyr701) (58D6) Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded Non-Hodgkin’s lymphoma using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167 in the presence of control peptide (left) or Phospho-Stat1 (Tyr701) Blocking Peptide (right).


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded stomach (chronic gastritis), using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb on SignalSlide® Phospho-Stat1/3/5 IHC Controls #8105 (paraffin-embedded HeLa cell pellets, untreated (left), treated with Human Interferon-α1 (hIFN-α1) #8927 (middle), or treated with Human Epidermal Growth Factor (hEGF) #8916 (right).

IHC-F (frozen)

IHC-F (frozen)

Immunohistochemical analysis of frozen SKOV-3 xenograft using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb.


Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HeLa cells, untreated (blue) or IFN-α treated (green), using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb compared to a nonspecific negative control antibody (red).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or IFNα-treated #9906 (right), using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Chromatin IP

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 5 μl of Phospho-Stat1 (Tyr701) (58D6) 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 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.


Background

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.

Application References

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Companion Products

Rabbit Monoclonals Produced Using Epitomics® Technology, U.S. Patent No. 5,675,063.

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

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