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9914
Phospho-Stat Antibody Sampler Kit

Phospho-Stat Antibody Sampler Kit #9914

Western Blotting Image 1

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).

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Chromatin IP-seq Image 2

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 from 5 ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. The figure shows binding across IRF1, 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.

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Western Blotting Image 3

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).

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Western Blotting Image 4

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.

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Western Blotting Image 5

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).

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Western Blotting Image 6

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.

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Western Blotting Image 7

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).

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Western Blotting Image 8

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 Cytometry Image 9

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

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Chromatin IP Image 10

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.

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IHC-P (paraffin) Image 11

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

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Chromatin IP Image 12

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) (D3A7) XP® 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 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.

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Chromatin IP Image 13

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, 30 min), and either 10 μl of Phospho-Stat3 (Ser727) 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.

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Flow Cytometry Image 14

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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Flow Cytometry Image 15

Flow cytometric analysis of Ramos cells, untreated (blue) or IL-4 treated (green), using Phospho-Stat6 (Tyr641) Antibody compared to a nonspecific negative control antibody (red).

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IF-IC Image 16

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).

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IHC-P (paraffin) Image 17

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.

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IF-IC Image 18

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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IF-IC Image 19

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).

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IHC-P (paraffin) Image 20

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

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IHC-P (paraffin) Image 21

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)).

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IHC-F (frozen) Image 22

Immunohistochemical analysis of frozen H1650 xenograft using Phospho-Stat3 (Tyr705)(D3A7) XP® Rabbit mAb.

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Flow Cytometry Image 23

Flow cytometric analysis of HeLa cells, untreated (blue) or IFN-α treated (green), using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

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IF-IC Image 24

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

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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
  • F
H 110 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

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.

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

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.

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, J.E. et al. (1994) Science 264, 1415-21.
  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, 241-50.
  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, 2151-9.
  10. Cattaneo, E. et al. (1999) Trends Neurosci. 22, 365-369.
  11. Frank, D.A. (1999) Mol. Med. 5, 432-456.
Entrez-Gene Id
6772 , 6773 , 6774 , 6776 , 6777 , 6778
Swiss-Prot Acc.
P42224 , P52630 , P40763 , P42229 , P51692 , P42226
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.

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