Cell Signaling Technology

Product Pathways - PI3K / Akt Signaling

PhosphoPlus® Akt (Ser473) Antibody Kit #9270

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 100 µl W IP IHC-P IHC-F IF-IC F H M R Hm Mk Dm Z B (C) (X) (Dg) (Pg) 60 Rabbit IgG
Akt (pan) (C67E7) Rabbit mAb #4691 100 µl W IP IHC-P IF-IC F H M R Mk Dm (Pg) 60 Rabbit IgG
Akt Control Cell Extracts #9273 200 µl
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat
Anti-biotin, HRP-linked Antibody #7075 100 µl Goat
Biotinylated Protein Ladder Detection Pack #7727 100 µl
20X LumiGLO® Reagent and 20X Peroxide #7003 5 ml each

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IHC-F=Immunohistochemistry (Frozen)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  C=Chicken  Dm=D. melanogaster  X=Xenopus  Z=Zebrafish  B=Bovine  Dg=Dog  Pg=Pig
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Phospho-Akt (Ser473) Antibody detects endogenous levels of Akt1 only when phosphorylated at Ser473, and Akt2 and Akt3 when phosphorylated at equivalent sites. It does not recognize Akt phosphorylated at other sites, nor does it recognize phosphorylated forms of related kinases such as PKC or p70 S6 kinase. Akt Antibody detects endogenous levels of total Akt1, Akt2 and Akt3 proteins. It does not cross-react with related kinases.

Western Blotting

Western Blotting

Western blot analysis of extracts from PC3 cells, untreated or LY294002/wortmannin-treated, and NIH/3T3 cells, serum- starved or PDGF-treated, using Phospho-Akt (Ser473) (D9E) Rabbit mAb #4060.

Western Blotting

Western Blotting

Western blot analysis of recombinant Akt1, Akt2 and Akt3 proteins, and extracts from various cell lines, using Akt (pan) (C67E7) Rabbit mAb #4691.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002, wortmannin and U0126 (blue), using Phospho-Akt (Ser473) (D9E) Rabbit mAb #4060 compared to a nonspecific negative control antibody (red).


Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Jurkat cells using Akt (pan) (C67E7) Rabbit mAb #4691 (blue) compared to a nonspecific negative control antibody (red).

Description

The PhosphoPlus® Akt (Ser473) Antibody Kit provides reagents and protocols for the rapid analysis of Akt phosphorylation.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser473 or the carboxy-terminal sequence of Akt.

Background

Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function 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 by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).

  1. Franke, T.F. et al. (1997) Cell 88, 435-7.
  2. Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.
  3. Franke, T.F. et al. (1995) Cell 81, 727-36.
  4. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
  5. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
  6. Jacinto, E. et al. (2006) Cell 127, 125-37.
  7. Cardone, M.H. et al. (1998) Science 282, 1318-21.
  8. Brunet, A. et al. (1999) Cell 96, 857-68.
  9. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
  10. Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.
  11. Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.
  12. Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.
  13. Cross, D.A. et al. (1995) Nature 378, 785-9.
  14. Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.
  15. Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30.
  16. Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52.
  17. Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
  18. Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57.
  19. Manning, B.D. et al. (2002) Mol Cell 10, 151-62.

Application References

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Protocols

* Product-specific protocol.

Companion Products

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

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