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 Kit #7100

Kit Includes Quantity Applications Reactivity MW (kDa) Source
PathScan® Multiplex Western Cocktail I: Phospho-p90RSK, Phospho-Akt, Phospho-p44/42 MAPK (Erk1/2) and Phospho-S6 Ribosomal Protein Detection Cocktail I # 5301 250 microliters W H M R 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  M=Mouse  R=Rat

Specificity / Sensitivity

Each phospho-antibody in this kit recognizes only the phosphorylated form of its specific target. The eIF4E antibody detects total levels of target protein to control for protein loading. All the antibodies detect endogenous levels of target proteins.

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.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with synthetic peptides. 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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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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