Cell Signaling Technology

Product Pathways - Screening Technologies

PathScan® Multiplex Western Cocktail II: Phospho-p90RSK, Phospho-p53, Phospho-p38 MAPK and Phospho-S6 Ribosomal Protein Detection Cocktail II #5302

Applications Reactivity Source
W H M R Mi Rabbit

Applications Key:  W=Western Blotting
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mi=Mink
Species cross-reactivity is determined by Western blot.

This Cocktail Contains The Following Antibodies:

Western Blotting

Western Blotting

Western blot analysis of extracts from Mv1Lu mink lung epithelial cells untreated or treated with UV and TPA, using PathScan Multiplex Western cocktail II to detect phosphorylation of p90RSK, p53, p38 MAPK and S6 ribosomal protein.

Description

The PathScan® Multiplex Western Cocktail II offers a unique method to assay the activation of multiple pathways on one membrane without stripping and reprobing. This method saves the user valuable time, while increasing accuracy and minimizing reagent waste. The system allows the user to simultaneously detect levels of phospho-p90RSK, phospho-p53, phospho-p38 MAPK and phospho-S6 ribosomal protein. The kit also includes elF4E antibody to control protein loading.

Specificity / Sensitivity

Each phospho-antibody in this cocktail recognizes endogenous levels of only the phosphorylated form of its specific target. The eIF4E antibody detects endogenous levels of its target protein independent of phosphorylation and is provided to control for protein loading.

Source / Purification

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

Background

The 90 kDa ribosomal S6 kinases (RSK1-3) are a family of serine/threonine kinases broadly expressed in response to many growth factors, polypeptide hormones and neurotransmitters (1). p90RSK is activated by Erk1 and Erk2 in vitro and in vivo via phosphorylation (2). Several sites, such as Ser380, Thr359 and Ser363, are important for its activation (3).The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis (4). p53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro (5,6). DNA damage induces phosphorylation of p53 at Ser15 and Ser20 and leads to reduced interaction of p53 with its negative regulator, oncoprotein MDM2 (7).p38 MAP kinase controls cellular responses to cytokines and stress (8-11). Like the SAPK/JNK pathway, p38 MAP kinase is activated by a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharides (LPS), UV light and growth factors (8-12). MKK3, MKK6 and SEK activate p38 MAP kinase by phosphorylation at Thr180 and Tyr182.Growth factors and mitogens induce the activation of p70 S6 kinase, which in turn phosphorylates 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 (13). 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 (13,14).

  1. Frodin, M. and Gammeltoft, S. (1999) Mol. Cell. Endocrinol. 151, 65-77.
  2. Lazar, D.F. et al. (1995) J. Biol. Chem. 270, 20801-20807.
  3. Dalby, K.N. et al. (1998) J. Biol. Chem. 273, 1496-1505.
  4. Levine, A.J. (1997) Cell 88, 323-331.
  5. Meek, D.W. (1994) Semin. Cancer Biol. 5, 203-210.
  6. Milczarek, G.J. et al. (1997) Life Sci. 60, 1-11.
  7. Shieh, S.Y. et al. (1997) Cell 91, 325-334.
  8. Han, J. et al. (1994) Science 265, 808-811.
  9. Lee, J.C. et al. (1994) Nature 372, 739-746.
  10. Rouse, J. et al. (1994) Cell 78, 1027-1037.
  11. Freshney, N.W. et al. (1994) Cell 78, 1039-1049.
  12. Raingeaud, J. et al. (1995) J. Biol. Chem. 270, 7420-7426.
  13. Peterson, R.T. and Schreiber, S.L. (1998) Curr. Biol. 8, R248-R250.
  14. Jefferies, H.B. et al. (1997) EMBO J. 16, 3693-3704.

Application References

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This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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