Cell Signaling Technology

Product Pathways - PathScan Multiplex WB Cocktails

PathScan® Multiplex Western Cocktail I: Phospho-p90RSK, Phospho-Akt, Phospho-p44/42 MAPK (Erk1/2) and Phospho-S6 Ribosomal Protein Detection Cocktail I #5301

Applications Reactivity Sensitivity Source
W H M R Endogenous Rabbit

Applications Key:  W=Western Blotting
Reactivity Key:  H=Human  M=Mouse  R=Rat
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

This Cocktail Contains The Following Antibodies:

Western Blotting

Western Blotting

Western blot analysis of extracts from CHO cells, untreated or insulin-treated following pretreatment with wortmannin (PI3 kinase inhibitor), rapamycin (mTOR inhibitor) and/or U0126 (MEK inhibitor) as indicated, using PathScan® Multiplex Western Cocktail I to detect phosphorylation of p90RSK, Akt, p44/42 MAPK and S6 ribosomal protein.

Description

The PathScan® Multiplex Western Cocktail I 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-Akt, phospho-p44/42 MAPK (Erk1/2) and phospho-S6 ribosomal protein. The cocktail also includes the Rab11 Antibody to control for protein loading.

Specificity / Sensitivity

Each phospho-antibody in this cocktail recognizes endogenous levels of only the phosphorylated form of its specific target. The Rab11 antibody detects endogenous levels of its target protein independent of phosphorylation, and is provided to control for protein loading.

Source / Purification

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

Background

Akt, also referred to as PKB or Rac, plays a critical role in controlling the balance between survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors, and functions in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and also by phosphorylation within the carboxy-terminus at Ser473.Both p44 and p42 MAP kinases (Erk1 and Erk2) play a critical role in the regulation of cell growth and differentiation (5-8). MAP kinases are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. Activation of MAP kinases occurs through phosphorylation of threonine and tyrosine (202 and 204 of human MAP kinase [Erk1] or 183 and 185 of rat Erk2) at the sequence T*EY* by a single upstream MAP kinase kinase (MEK) (9,10). One of the downstream targets of p44/42 MAPK is p90RSK.To effectively promote growth and cell division in a sustained manner, growth factors and mitogens must upregulate translation (11,12). 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 (12).

  1. Franke, T.F. (1997) Cell 88, 435-437.
  2. Burgering, B.T. and Coffer, P.J. (1995) Nature 376, 599-602.
  3. Franke, T. F. et al. (1995) Cell 81, 727-736.
  4. Alessi, D.R. et al. (1996) EMBO J. 15, 6541-6551.
  5. Marshall, C.J. (1995) Cell 80, 179-185.
  6. Hunter, T. (1995) Cell 80, 225-236.
  7. Hill, C.S. and Treisman, R. (1995) Cell 80, 199-211.
  8. Cowley, S. et al. (1994) Cell 77, 841-852.
  9. Sturgill, T.W. et al. (1988) Nature 334, 715-718.
  10. Payne, D. M. et al. (1991) EMBO J. 10, 885-892.
  11. Dufner, A. and Thomas, G. (1999) Exp. Cell. Res. 253, 100-109.
  12. Peterson, R.T. and Schreiber, S.L. (1998) Curr. Biol. 8, R248-R250.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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