Product Pathways - PI3K / Akt Signaling
Phospho-Akt (Ser473) Antibody #9271
|9271L||300 µl (30 western blots)||---||In Stock||---|
|9271S||100 µl (10 western blots)||---||In Stock||---|
|9271||carrier free and custom formulation / quantity||email request|
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|W||1:1000||Human, Mouse, Rat, Hamster, D. melanogaster, Bovine, Dog, Pig||Endogenous||60||Rabbit|
Species cross-reactivity is determined by western blot.
Applications Key: W=Western Blotting, IP=Immunoprecipitation, IF-IC=Immunofluorescence (Immunocytochemistry), F=Flow Cytometry
Species predicted to react based on 100% sequence homology: Monkey, Chicken, Xenopus, Horse.
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 blot analysis of extracts from NIH/3T3 cells, untreated or treated with PDGF, wortmannin, LY294002, rapamycin or PD98059, using Phospho-Akt (Ser473) Antibody.
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 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.  J. Biol. Chem. 273, 23534-23541.)
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).
Confocal immunofluorescent analysis of C2C12 cells, treated with LY294002 #9901 (50 uM, 2 hrs; left) or insulin-treated (100 ng/mL, 30 min; right), using Phospho-Akt (Ser473) Antibody (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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).
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- Patrucco, E. et al. (2004) Cell 118, 375-387. Applications: Western Blotting.
- Fukuda, T. et al. (2003) J. Biol. Chem. 278, 51324-51333. Applications: Western Blotting.
- Okkenhaug, K. et al. (2002) . Science 297 (5583), 1031-1034. Applications: Western Blotting.
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- Fujita, H. et al. (2005) Biochem Pharmacol 69, 1773-84. Applications: Western Blotting.
- Engelman, J.A. et al. (2008) Nat Med , . Applications: Western Blotting.
- Hoshino, Y. et al. (2004) Mol Reprod Dev 69, 77-86. Applications: IF-IC (In Cells).
- Chung, Y.W. et al. (2011) J Biol Chem 286, 29681-90. Applications: Western Blotting.
- Fonseca, B.D. et al. (2011) J Biol Chem 286, 27111-22. Applications: Western Blotting.
- Kubota, H. et al. (2012) Mol Cell 46, 820-32. Applications: Western Blotting.
- Zhang, J. et al. (2013) Nat Cell Biol , . Applications: Western Blotting.
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