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Western blot analysis of extracts from UT-7 cells, untreated or treated with erythropoietin (EPO; 3 units/ml for 5 min), TF-1 cells, untreated or treated with Human Granulocyte Macrophage Colony Stimulating Factor #8922 (hGM-CSF; 100ng/ml for 10 min), and NK-92 cells, untreated or treated with Human Interleukin-2 #8907 (hIL-2; 100ng/ml for 10 min), using Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb (upper) or total Stat5 (3H7) Rabbit mAb #9358 (lower).

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Western blot analysis of extracts from various cell lines using the Stat5 Antibody.

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After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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Flow cytometric analysis of TF-1 cells, untreated (blue) or GM-CSF treated (green), using Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb.

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Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 BaF3 cells starved of IL-3 for 6 hours followed by induction with IL-3 for 45 minutes, and either 20 μl of Stat5 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 SimpleChIP® Mouse CIS Intron 1 Primers #5131, mouse SOCS3 promoter primers, and SimpleChIP® Mouse RPL30 Intron 2 Primers #7015. 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.

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Confocal immunofluorescent analysis of A-431 cells, EGF-treated (left) or untreated (right), using Phospho-Stat5 (Tyr694) XP®(D47E7) Rabbit mAb (green) and Pan-Keratin (C11) Mouse mAb #4545 (red).

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Image
Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb 4322 100 µl
Western Blotting Immunofluorescence Flow Cytometry
H M 90 Rabbit IgG
Stat5 Antibody 9363 100 µl
Western Blotting Immunoprecipitation Chromatin Immunoprecipitation
H M R Mk 90 Rabbit 
Stat5 Control Cell Extracts 9353 100 µl
Human 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
Western Blotting
All Goat 
Anti-biotin, HRP-linked Antibody 7075 100 µl
Goat 
Biotinylated Protein Ladder Detection Pack 7727 100 µl
 
20X LumiGLO® Reagent and 20X Peroxide 7003 5 ml each
Western Blotting
 

Product Description

The PhosphoPlus® Stat5 (Tyr694) Antibody Kit provides reagents and protocols for the rapid analysis of the phosphorylation status of Stat5a at Tyr694 and Stat5b at Tyr699.


Specificity / Sensitivity

Phospho-Stat5 (Tyr694) (D47E7) XPTM Rabbit mAb detects endogenous levels of Stat5a only when phosphorylated at Tyr694 and Stat5b when phosphorylated at Tyr699. Stat5 (3H7) Rabbit mAb detects endogenous levels of total Stat5α and ß protein.


Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr694 of human Stat5a protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to an amino-terminal region within Stat5. Antibodies were purified by protein A and peptide affinity chromatography.

Stat5 is activated in response to a wide variety of ligands including IL-2, GM-CSF, growth hormone and prolactin. Phosphorylation at Tyr694 is obligatory for Stat5 activation (1,2). This phosphorylation is mediated by Src upon erythropoietin stimulation (3). Stat5 is constitutively active in some leukemic cell types (4). Phosphorylated Stat5 is found in some endothelial cells treated with IL-3, which suggests its involvement in angiogenesis and cell motility (5). Stat5a and Stat5b are independently regulated and activated in various cell types. For instance, interferon treatment predominantly activates Stat5a in U-937 cells and Stat5b in HeLa cells (6).


1.  Gouilleux, F. et al. (1994) EMBO J 13, 4361-9.

2.  Wakao, H. et al. (1994) EMBO J 13, 2182-91.

3.  Okutani, Y. et al. (2001) Oncogene 20, 6643-50.

4.  Demoulin, J.B. et al. (1999) J Biol Chem 274, 25855-61.

5.  Dentelli, P. et al. (1999) J Immunol 163, 2151-9.

6.  Meinke, A. et al. (1996) Mol Cell Biol 16, 6937-44.


Entrez-Gene Id 6776, 6777
Swiss-Prot Acc. P42229, P51692


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
PhosphoPlus® is a trademark of Cell Signaling Technology, Inc.
LumiGLO® is a registered trademark of Kirkegaard & Perry Laboratories.