Revision 1

#9914Store at -20C

1 Kit

(6 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb 7649 20 µl 84, 91 kDa Rabbit IgG
Phospho-Stat2 (Tyr690) Antibody 4441 20 µl 113 kDa Rabbit 
Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb 9145 20 µl 79, 86 kDa Rabbit IgG
Phospho-Stat3 (Ser727) Antibody 9134 20 µl 86 kDa Rabbit 
Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb 4322 20 µl 90 kDa Rabbit IgG
Phospho-Stat6 (Tyr641) Antibody 9361 20 µl 110 kDa Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

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.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

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.

Background References

    Trademarks and Patents

    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.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #9914

    Phospho-Stat Antibody Sampler Kit

    Phospho-Stat Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from serum-starved NK-92 cells treated with hIL-2 (10 ng/mL, O/N) using Phospho-Stat5 (Tyr694) (D47E7) XP® Rabbit mAb #4322. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Phospho-Stat Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from HeLa cells, untreated or treated with IFNa (#36000, 100 ng/mL, 5 min) or IFNg (#80385, 100 ng/mL, 30 min); using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb #9145 (upper), Stat3 (D3Z2G) Rabbit mAb #12640 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Phospho-Stat Antibody Sampler Kit: Image 3 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from serum-starved HeLa cells treated with IFN-alpha (100 ng/mL, 5 min) using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb #9145. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Phospho-Stat Antibody Sampler Kit: Image 4 Expand Image
    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 5 Expand Image
    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 6 Expand Image
    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 7 Expand Image
    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 8 Expand Image
    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 9 Expand Image
    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.
    Phospho-Stat Antibody Sampler Kit: Image 10 Expand Image
    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 11 Expand Image
    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 Expand Image
    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 13 Expand Image
    Immunoprecipitation of phospho-Stat3 (Tyr705) from U266 extracts treated with human IFN-α (50 ng/ml, 15 min). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb. Western blot analysis was performed using Phospho-Stat3 (Tyr705) (3E2) Mouse mAb #9138. Anti-mouse IgG, HRP-linked Antibody #7076 was used as a secondary antibody.
    Phospho-Stat Antibody Sampler Kit: Image 14 Expand Image
    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 15 Expand Image
    Flow cytometric analysis of TF-1 cells, untreated (blue, low) or treated with hGM-CSF #8922 (50 ng/ml, 15 min; green, positive) using Phospho-Stat5 (Tyr694) (D47E7) 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 16 Expand Image
    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 Expand Image
    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 18 Expand Image
    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 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #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 datasheet.
    Phospho-Stat Antibody Sampler Kit: Image 19 Expand Image
    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 20 Expand Image
    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 21 Expand Image
    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 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #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 22 Expand Image
    Immunohistochemical analysis of paraffin-embedded Apc (min/+) mouse intestine, using Phosho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.
    Phospho-Stat Antibody Sampler Kit: Image 23 Expand Image
    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 24 Expand Image
    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 25 Expand Image
    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 26 Expand Image
    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 Expand Image
    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 28 Expand Image
    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 29 Expand Image
    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 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #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 datasheet.
    Phospho-Stat Antibody Sampler Kit: Image 30 Expand Image
    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 31 Expand Image
    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.
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.