PI3K / Akt Signaling

Phospho-Akt (Ser473) (D9E) XP™ Rabbit mAb (HRP Conjugate) #5171

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Applications Key:   W = Western Blotting
Reactivity Key: B = Bovine  Dm = D. melanogaster  H = Human  Hm = Hamster  M = Mouse  Mk = Monkey  R = Rat  Z = Zebrafish  
Species cross-reactivity is determined by Western blot.

Protocols

5171

Western Blotting,

Specificity / Sensitivity

Phospho-Akt (Ser473) (D9E) XP™ Rabbit mAb (HRP Conjugate) detects endogenous levels of Akt only when phosphorylated at Ser473.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser473 of human Akt protein.

Description

This Cell Signaling Technology antibody is conjugated to the carbohydrate groups of horseradish peroxidase (HRP) via its amine groups. The HRP conjugated antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-Akt (Ser473) (D9E) XP™ Rabbit mAb #4060.

Western Blotting

Western blot analysis of extracts from NIH/3T3 and Jurkat cells, untreated or treated with either PDGF, Calyculin A or LY294002 #9901 as indicated, using Phospho-Akt (Ser473) (D9E) XP™ Rabbit mAb (HRP Conjugate).

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). Inhibition of mTOR stops the protein synthesis machinery by inactivating p70 S6 kinase and activating the eukaryotic initiation factor 4E binding protein 1 (4E-BP1), an inhibitor of translation (18,19).

1. Franke, T.F. et al. (1997) Cell88, 435 - 7.
2. Burgering, B.M. and Coffer, P.J. et al. (1995) Nature376, 599 - 602.
3. Franke, T.F. et al. (1995) Cell81, 727 - 36.
4. Alessi, D.R. et al. (1996) EMBO J15, 6541 - 51.
5. Sarbassov, D.D. et al. (2005) Science307, 1098 - 101.
6. Jacinto, E. et al. (2006) Cell127, 125 - 37.
7. Cardone, M.H. et al. (1998) Science282, 1318 - 21.
8. Brunet, A. et al. (1999) Cell96, 857 - 68.
9. Zimmermann, S. and Moelling, K. et al. (1999) Science286, 1741 - 4.
10. Cantley, L.C. and Neel, B.G. et al. (1999) Proc Natl Acad Sci USA96, 4240 - 5.
11. Vlahos, C.J. et al. (1994) J Biol Chem269, 5241 - 8.
12. Hajduch, E. et al. (2001) FEBS Lett492, 199 - 203.
13. Cross, D.A. et al. (1995) Nature378, 785 - 9.
14. Diehl, J.A. et al. (1998) Genes Dev12, 3499 - 511.
15. Gesbert, F. et al. (2000) J Biol Chem275, 39223 - 30.
16. Zhou, B.P. et al. (2001) Nat Cell Biol3, 245 - 52.
17. Navé, B.T. et al. (1999) Biochem J344 Pt 2, 427 - 31.
18. Inoki, K. et al. (2002) Nat Cell Biol4, 648 - 57.
19. Manning, B.D. et al. (2002) Mol Cell10, 151 - 62.

Application References

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Companion Products

• 2920 Akt (pan) (40D4) Mouse mAb
• 4298 Akt (pan) (40D4) Mouse mAb (HRP Conjugate)
• 4348 p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (HRP Conjugate)
• 4060 Phospho-Akt (Ser473) (D9E) XP™ Rabbit mAb
• 5056 Phospho-Akt (Thr308) (C31E5E) Rabbit mAb (Biotinylated)
• 5012 Phospho-Akt (Ser473) (D9E) XP™ Rabbit mAb (Biotinylated)
• 2965 Phospho-Akt (Thr308) (C31E5E) Rabbit mAb
• 4051 Phospho-Akt (Ser473) (587F11) Mouse mAb
• 4691 Akt (pan) (C67E7) Rabbit mAb

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.