Cell Signaling Technology

Product Pathways - Akt Signaling

Phospho-Akt (Thr308) Blocking Peptide #1145

Description

This peptide can be used to specifically block Phospho-Akt (Thr308) (244F9) Rabbit mAb #4056 and Phospho-Akt (Thr308) Antibody #9275 reactivity.

Quality Control

The quality of the peptide was evaluated by reversed-phase HPLC and by mass spectrometry. The peptide blocks Phospho-Akt (Thr308) (244F9) Rabbit mAb #4056 signal in Western blotting and immunohistochemistry. It also blocks signal from and Phospho-Akt (Thr308) Antibody #9275 in Western blotting.

Western Blotting

Western Blotting

Western Blot analysis of extracts from NIH/3T3 cells, untreated or PDGF-treated, using Phospho-Akt (Thr308) (244F9) Rabbit mAb #4056 or the same antibody preincubated with Phospho-Akt (Thr308) Blocking Peptide.

Western Blotting

Western Blotting

Western Blot analysis of extracts from NIH/3T3 cells, untreated or PDGF-treated, using Phospho-Akt (Thr308) Antibody #9275 or the same antibody preincubated with Phospho-Akt (Thr308) Blocking Peptide.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-Akt (Thr308) (244F1) Rabbit mAb #4056 preincubated with an unrelated control peptide (left) or with Phospho-Akt (Thr308) Blocking Peptide (right).


Applications

Use as a blocking reagent to evaluate the specificity of antibody reactivity in Western immunoblotting and immunohistochemistry protocols.

Directions for Use

For immunohistochemistry, add twice the volume of peptide as volume of antibody used in 100 µl total volume. Incubate for a minimum of 30 minutes prior to adding the entire volume to the slide. Recommended antibody dilutions can be found on the relevant product data sheet.

For Western immunoblotting, add 10 µl of antibody and 10 µl of blocking peptide to 10 ml of antibody dilution buffer, and incubate at room temperature for 30 minutes before allowing to react with the blot.

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 by phosphorylating and inactivating 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 due to inactivation of its effector, p70 S6 kinase and activation of the eukaryotic initiation factor 4E binding protein 1 (4E-BP1), an inhibitor of translation (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. Nave, 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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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.

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