Cell Signaling Technology

Product Pathways - Jak/Stat Pathway

Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb #7649

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

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
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) (D4A7) Rabbit mAb recognizes endogenous levels of Stat1 protein only when phosphorylated at Tyr701.

Source / Purification

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

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa, A20, and PC-12 cells, untreated or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml, 30 min), using Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb (upper) or Stat1 Antibody #9172 (lower).

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with hIFN-α1 #8927 (green), using Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with hIFN-α1 #8927 (100 ng/mL, 30 min; right), using Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb (green) and β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red).


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) (D4A7) 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

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

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