Cell Signaling Technology

Product Pathways - PI3K / Akt Signaling

Phospho-Akt (Ser473) Antibody #9271

Applications Reactivity Sensitivity MW (kDa) Source
W IP IF-IC F H M R Hm Dm B Dg Pg (Mk) (C) (X) (Hr) Endogenous 60 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  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  B=Bovine  Dg=Dog  Pg=Pig  Hr=Horse
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Phospho-Akt (Ser473) Antibody detects endogenous levels of Akt1 only when phosphorylated at Ser473. This antibody also recognizes Akt2 and Akt3 when phosphorylated at the corresponding residues. 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.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser473 of mouse Akt. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from NIH/3T3 cells, untreated or treated with PDGF, wortmannin, LY294002, rapamycin or PD98059, using Phospho-Akt (Ser473) Antibody.

Western Blotting

Western Blotting

Western blot analysis of extracts from NIH/3T3 cells, untreated or treated with PDGF for the indicated times, using Phospho-Akt (Ser473) Antibody (upper) or Akt Antibody #9272 (lower).

Western Blotting

Western Blotting

Western blot analysis of immunoprecipitated Akt from 293 cells transiently transfected with HA-tagged Akt (WT), HA-tagged K179A mutant Akt and HA-tagged K179A/S473A mutant Akt, using Phospho-Akt (Ser473) Antibody (upper), Akt antibody (middle) or HA antibody (lower). Phospho-Akt (Ser473) Antibody does not recognize Akt with an alanine substituion at Ser473. (Polakiewicz, R.D. et al. [1998] J. Biol. Chem. 273, 23534-23541.)


Flow Cytometry

Flow Cytometry

Flow cytometric analysis of LNCaP cells, untreated (green) or LY294002-treated (blue), using Phospho-Akt (Ser473) Antibody compared to a nonspecific negative control antibody (red).

IF-IC

IF-IC

Confocal immunofluorescent images of C2C12 cells serum starved and treated with or without insulin as indicated and labeled with Phospho-Akt (Ser473) Antibody (red). Actin filaments have been labeled with fluorescein phalloidin.

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 Kip (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.

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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