Product Pathways - PI3K / Akt Signaling

SignalSilence^® Akt siRNA I #6211

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Description

SignalSilence^® Akt siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Akt expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence^® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.

Quality Control

Oligonucleotide synthesis is monitored base by base through trityl analysis to ensure appropriate coupling efficiency. The oligo is subsequently purified by affinity-solid phase extraction. The annealed RNA duplex is further analyzed by mass spectrometry to verify the exact composition of the duplex. Each lot is compared to the previous lot by mass spectrometry to ensure maximum lot-to-lot consistency.

Directions for Use

CST recommends transfection with 100 nM Akt siRNA I 48 to 72 hours prior to cell lysis. For transfection procedure, follow protocol provided by the transfection reagent manufacturer. Please fell free to contact CST with any questions on use.

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

Small interfering RNA (siRNA) has been used to specifically silence Akt in CHO cells and 3T3-L1 adipocytes (20).

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.
20. Katome, T. et al. (2003) J Biol Chem 278, 28312-23.

Application References

• Kakazu, A. et al. (2004) Invest. Ophthalmol. Vis. Sci. 45, 3485-92. Applications: Western Blotting siRNA
• Kierbel, A. et al. (2005) Mol. Biol. Cell 16, 2577-85. Applications: Western Blotting siRNA
• Wu, K.L. et al. (2004) J. Biol. Chem. 279, 26280-6. Applications: Western Blotting siRNA
• Chandrasekar, B. et al. (2005) J. Biol. Chem. 280, 4553-67. Applications: Western Blotting siRNA

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

• 6568 SignalSilence^® Control siRNA (Unconjugated)
• 6201 SignalSilence^® Control siRNA (Fluorescein Conjugate)
• 6510 SignalSilence^® Akt siRNA II
• 6511 SignalSilence^® Akt siRNA Kit
• 4685 Akt (pan) (11E7) Rabbit mAb

Limited Use Label License, RNA interference: This product is licensed under European Patent 1144623 and foreign equivalents from Ribopharma AG, Kulmbach, Germany and is provided only for use in non-commercial research specifically excluding use (a) in drug discovery or drug development, including target identification or target validation, by or on behalf of a commercial entity, (b) for contract research or commercial screening services, (c) for the production or manufacture of siRNA-related products for sale, or (d) for the generation of commercial databases for sale to Third Parties. Information about licenses for these and other commercial uses is available from Ribopharma AG, Fritz-Hornschuch-Str. 9, D-95326 Kulmbach, Germany.

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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.