Cell Signaling Technology

Product Pathways - PI3K / Akt Signaling

Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #43506

Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
F 1:50 Human,Mouse,Rat,Monkey, Endogenous Rabbit IgG
IF-IC 1:50

Species cross-reactivity is determined by western blot.

Applications Key: F=Flow Cytometry, IF-IC=Immunofluorescence (Immunocytochemistry),

Specificity / Sensitivity

Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of Akt1 protein only when phosphorylated at Thr308. This antibody also recognizes endogenous levels of Akt2 protein when phosphorylated at Thr309 or Akt3 protein when phosphorylated at Thr305.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr308 of human Akt1 protein.

Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometric (mouse cells) and immunofluorescence (human cells) analysis. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb #13038.

IF-IC

IF-IC

Confocal immunofluorescent analysis of MCF7 cells, either treated with LY294002 #9901 (50 μM, 2hrs; left) or treated with insulin (100 nM, 15 min; right), using Phospho-Akt (Thr308) (D25E7) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of serum-starved NIH/3T3 cells, untreated (blue) or treated with mouse platelet-derived growth factor BB (200 ng/ml, 15 min; green), using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb (Alexa Fluor® 488 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 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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For Research Use Only. Not For Use In Diagnostic Procedures.

DRAQ5 is a registered trademark of Biostatus Limited.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

XP is a registered trademark of Cell Signaling Technology, Inc.

The Alexa Fluor dye conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS).

This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchased product solely in research, including use with HCS or other automated imaging applications but excluding use in combination with DNA microarrays. The buyer must not sell or otherwise transfer this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; (c) manufacturing or quality assurance or quality control, or (d) resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.

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