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9914
Phospho-Stat Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Phospho-Stat Antibody Sampler Kit #9914

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Phospho-Stat Antibody Sampler Kit: Image 1

Chromatin immunoprecipitations were performed with cross-linked chromatin from HT-1080 cells treated with IFN-γ (50 ng/ml) for 30 minutes and Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across IRF-1, a known target gene of Phospho-Stat1 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

Phospho-Stat Antibody Sampler Kit: Image 2

Western blot analysis of extracts from HeLa cells, untreated or IFN-α-treated (100 ng/ml, 15 minutes), using Stat2 Antibody (upper) or Phospho-Stat2 (Tyr690) Antibody (lower).

Phospho-Stat Antibody Sampler Kit: Image 3

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hep G2 cells starved overnight and treated with IL-6 (100 ng/ml) for 30 minutes and Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across IRF1, a known target gene of Phospho-Sata3 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

Phospho-Stat Antibody Sampler Kit: Image 4

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hep G2 cells starved overnight and treated with IL-6 (100 ng/ml, 30 min), and either Phospho-Stat3 (Ser727) Antibody or 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.

Phospho-Stat Antibody Sampler Kit: Image 5

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

Phospho-Stat Antibody Sampler Kit: Image 6

Confocal immunofluorescent analysis of ACHN cells serum-starved (left) or IL-4-treated (right) using Phospho-Stat6 (Tyr641) Antibody (green). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).

Phospho-Stat Antibody Sampler Kit: Image 7

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.

Phospho-Stat Antibody Sampler Kit: Image 8

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 5 μl of Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 5 (upper), including IRF1 (lower), a known target gene of Phospho-Stat1 (see additional figure containing ChIP-qPCR data).

Phospho-Stat Antibody Sampler Kit: Image 9

Chromatin immunoprecipitations were performed with cross-linked chromatin from HaCaT cells Hep G2 cells starved overnight and treated with IL-6 (100 ng/ml) for 30 minutes and either Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Sonication Chromatin IP Kit #56383. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human c-Fos Promoter Primers #4663, human IRF1 promoter primers, 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.

Phospho-Stat Antibody Sampler Kit: Image 10

Western blot analysis of extracts from A172 cells, untreated (-) or UV-treated (100 mJ, 30 min; +) with or without λ phosphatase (+), using Phospho-Stat3 (Ser727) Antibody (upper) and Stat3 (79D7) Rabbit mAb #4904 (lower).

Phospho-Stat Antibody Sampler Kit: Image 11

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).

Phospho-Stat Antibody Sampler Kit: Image 12

Western blot analysis of extracts from Daudi cells, treated with IL-4 (100 ng/ml) for the indicated times using Phospho-Stat6 (Tyr641) Antibody (upper) or Stat6 antibody (lower).

Phospho-Stat Antibody Sampler Kit: Image 13

Chromatin immunoprecipitations were performed with cross-linked chromatin from HT-1080 cells treated with IFN-γ (50 ng/ml) for 30 minutes and either Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb or 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.

Phospho-Stat Antibody Sampler Kit: Image 14

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hep G2 cells starved overnight and treated with IL-6 (100 ng/ml) for 30 minutes, and either Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb or 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.

Phospho-Stat Antibody Sampler Kit: Image 15

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; 100 ng/ml for 10 min), and NK-92 cells, untreated or treated with Human Interleukin-2 #8907 (hIL-2; 100 ng/ml for 10 min), using Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb.

Phospho-Stat Antibody Sampler Kit: Image 16

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

Phospho-Stat Antibody Sampler Kit: Image 17

Flow cytometric analysis of U266 cells, untreated (blue) or treated with IFNalpha (50 ng/ml, 15 min; green) using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (solid lines) or concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Phospho-Stat Antibody Sampler Kit: Image 18

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).

Phospho-Stat Antibody Sampler Kit: Image 19

Confocal immunofluorescent analysis of HeLa cells, IFN-alpha treated (left) or untreated (right), labeled with Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (green).

Phospho-Stat Antibody Sampler Kit: Image 20

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).

Phospho-Stat Antibody Sampler Kit: Image 21

Immunohistochemical analysis of paraffin-embedded human neuroendocrine lung carcinoma using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb performed on the Leica® BOND™ Rx.

Phospho-Stat Antibody Sampler Kit: Image 22

Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb performed on the Leica® BOND™ Rx.

Phospho-Stat Antibody Sampler Kit: Image 23

Immunohistochemical analysis of paraffin-embedded Apc (min/+) mouse intestine, using Phosho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

Phospho-Stat Antibody Sampler Kit: Image 24

Immunohistochemical analysis of paraffin embedded human breast carcinoma, specifically endothelial cells, untreated (left) or lambda phosphatase treated (right), using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

Phospho-Stat Antibody Sampler Kit: Image 25

Immunnohistochemical analysis of paraffin-embedded human lung carcinoma, showing nuclear localization, using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

Phospho-Stat Antibody Sampler Kit: Image 26

Immunohistochemical analysis using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb on SignalSlide® HeLa -/+ IFNa IHC Controls #55861 (paraffin-embedded HeLa cell pellets, untreated (left) or treated with Human Interferon-α1 (hIFN-α1) #8927 (right)).

Phospho-Stat Antibody Sampler Kit: Image 27

Western blot analysis of extracts from IFN-alpha treated Jurkat cells and HeLa cells (left), as well as EGF treated A431 cells (right), using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb. Note that the basal phospho-Stat3 in A431 is detected by the antibody.

To Purchase # 9914T
Product # Size Price
9914T
1 Kit  (6 x 20 µl) $ 445

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb 7649 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R 84, 91 Rabbit IgG
Phospho-Stat2 (Tyr690) Antibody 4441 20 µl
  • WB
H 113 Rabbit 
Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb 9145 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H M R Mk 79, 86 Rabbit IgG
Phospho-Stat3 (Ser727) Antibody 9134 20 µl
  • WB
  • IP
  • ChIP
H M R 86 Rabbit 
Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb 4322 20 µl
  • WB
  • IF
  • F
H M 90 Rabbit IgG
Phospho-Stat6 (Tyr641) Antibody 9361 20 µl
  • WB
  • IP
  • IF
H 110 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The Phospho-Stat Pathway Sampler Kit provides an economical means to evaluate the activation status of Stat molecules, including the phosphorylation of Stat1 at Tyr701, Stat2 at Tyr690, Stat3 at Tyr705/Ser727, Stat5 at Tyr694 and Stat6 at Tyr641. The kit includes enough primary and secondary antibody to perform two Western blot experiments.

Specificity / Sensitivity

Each phospho-Stat antibody in the kit recognizes only the phosphorylated form of its specific target.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptides corresponding to residues surrounding Tyr690 of human Stat2, Ser727 of mouse Stat3, or Tyr641 of human Stat6. Antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr701 of human Stat1 protein, Tyr705 of mouse Stat3, or residues surrounding Tyr694 of human Stat5a protein.

Background

Jaks (Janus Kinases) and Stats (Signal Transducers and Activators of Transcription) are utilized by receptors for a wide variety of ligands including cytokines, hormones, growth factors and neurotransmitters. Jaks, activated via autophosphorylation following ligand-induced receptor aggregation, phosphorylate tyrosine residues on associated receptors, Stat molecules and other downstream signaling proteins (1,2). The phosphorylation of Stat proteins at conserved tyrosine residues activates SH2-mediated dimerization followed rapidly by nuclear translocation. Stat dimers bind to IRE (interferon response element) and GAS (gamma interferon-activated sequence) DNA elements, resulting in the transcriptional regulation of downstream genes (1,2). The remarkable range and specificity of responses regulated by the Stats is determined in part by the tissue-specific expression of different cytokine receptors, Jaks and Stats (2,3), and by the combinatorial coupling of various Stat members to different receptors. Serine phosphorylation in the carboxy-terminal transcriptional activation domain has been shown to regulate the function of Stat1, -2, -3, -4 and -5 (1). Phosphorylation of Stat3 at Ser727 via MAPK or mTOR pathways is required for optimal transcriptional activation in response to growth factors and cytokines including IFN-gamma and CNTF (4,5). Jak/Stat pathways also play important roles in oncogenesis, tumor progression, angiogenesis, cell motility, immune responses and stem cell differentiation (6-11).

  1. Darnell Jr., J. et al. (1994) Science 264, 1415-1421.
  2. Leonard, W.J. and O'Shea, J.J. (1998) Annu. Rev. Immunol. 16, 293-322.
  3. Caldenhoven, E. et al. (1996) J. Biol. Chem. 271, 13221-13227.
  4. Wen, Z. et al. (1995) Cell 82, 241-250.
  5. Yokogami, K. et al. (2000) Curr. Biol. 10, 47-50.
  6. Lim, C.P. and Cao, X. (1999) J. Biol. Chem. 274, 31055-31061.
  7. Bromberg, J. F. et al. (1999) Cell 98, 295-303.
  8. Su, L. et al. (1999) J. Biol. Chem. 274, 31770-31774.
  9. Dentelli, P. et al. (1999) J. Immunol. 163, 2151-2159.
  10. Cattaneo, E. et al. (1999) Trends Neurosci. 22, 365-369.
  11. Frank, D.A. (1999) Mol. Med. 5, 432-456.

Pathways & Proteins

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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.