Cell Signaling Technology

Product Pathways - PathScan Multiplex WB Cocktails

PathScan® Multiplex Western Cocktail III: Phospho-Stat1, Phospho-SAPK/JNK, Phospho-S6 Ribosomal Protein and Phospho-HSP27 Detection Kit #7120

Kit Includes Quantity Applications Reactivity MW (kDa) Source
PathScan® Multiplex Western Cocktail III: Phospho-Stat1, Phospho-SAPK/JNK, Phospho-S6 Ribosomal Protein and Phospho-HSP27 Detection Cocktail III # 5303 250 microliters W H Rabbit
Treated and Untreated Control Cell Extracts 50 microliters
Anti-rabbit IgG, HRP-linked Antibody # 7074 25 microliters Goat
Anti-biotin, HRP-linked Antibody # 7075 50 microliters Goat
20X LumiGLO® Reagent and 20X Peroxide # 7003 2.5 milliliters
Biotinylated Protein Ladder Detection Pack # 7727 50 microliters

Applications Key:  W=Western Blotting
Reactivity Key:  H=Human

Specificity / Sensitivity

Each phospho-antibody in this kit recognizes only the phosphorylated form of its specific target. The Pin1 antibody detects total levels of target protein to control for protein loading. All the antibodies in this kit detect endogenous levels of target proteins.

Western Blotting

Western Blotting

Western blot anlysis of extracts from HeLa cells, untreated or treated with IFN-alpha, UV and TPA, using PathScan® Multiplex Western Cocktail III to detect the phosphorylation of Stat1, SAPK/JNK, S6 ribosomal protein and HSP27.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with synthetic peptides. Antibodies are purified by protein A and peptide affinity chromatography.

Background

Stat1, while activated in response to a large number of ligands, appears to be essential for responsiveness to IFN-alpha and IFN-gamma (1-3).Phosphorylation of Stat1 at Tyr701 induces Stat1 dimerization, nuclear translocation and DNA binding (4). Stat1 has been found to be inappropriately activated in many tumors (5).The stress-activated protein kinase/Jun-terminal kinase SAPK/JNK is potently and preferentially activated by a variety of environmental stresses, including UV and gamma radiation, ceramides, inflammatory cytokines and, in some instances, by growth factors and GPCR agonists (6,7). SAPK/JNK, when active as a dimer, can translocate to the nucleus where it regulates transcription through its effects on c-Jun, ATF-2 and other transcription factors (8).To effectively promote growth and cell division in a sustained manner, growth factors and mitogens must upregulate translation (9,10). Growth factors and mitogens induce the activation of p70 S6 kinase, which in turn phosphorylate the S6 ribosomal protein. Phosphorylation of S6 correlates with an increase in translation, particularly of mRNAs with an oligopyrimidine tract in their 5' untranslated regions (10). This group of mRNAs (5'TOP) encodes proteins involved in cell cycle progression and proteins that are part of the translational machinery, such as ribosomal proteins and elongation factors (10,11).Heat shock protein (HSP) 27 is one of the small HSPs, regulated at both the transcriptional and posttranslational levels (12). In response to stress, the expression level of HSP27 increases several-fold to confer cellular resistance to the adverse environmental change. HSP27 is also phosphorylated at serines 15, 78 and 82 by MAPKAP kinase 2 as a result of p38 MAP kinase pathway activation (13,14).

  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. Davis, R.J. (1999) Biochem. Soc. Symp. 64, 1-12.
  7. Kyriakis, J.M. and Avruch, J. (2001) Phisiol. Rev. 81, 807-869.
  8. Whitmarsh, A.J. and Davis, R.J. (1998) Trends Biochem. Sci. 23, 481-485.
  9. Dufner, A. and Thomas, G. (1999) Exp. Cell. Res. 253, 100-109.
  10. Peterson, R.T. and Schreiber, S.L. (1998) Curr. Biol. 8, R248-R250.
  11. Jefferies, H.B. et al. (1997) EMBO J. 16, 3693-3704.
  12. Arrigo, A.P. and Landry, J. (1994) The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, NY 335-373.
  13. Landry, J. et al. (1992) J. Biol. Chem. 267, 794-803.
  14. Rouse, J. et al. (1994) Cell 78, 1027-1037.

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