Cell Signaling Technology

Product Pathways - Translational Control

SignalSilence® mTOR siRNA #6381

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, transfected with either control siRNA (-) or mTOR siRNA (+). mTOR was detected using mTOR Antibody #2972, and eIF4B was detected using eIF4B Antibody #3592. The mTOR Antibody confirms silencing of mTOR expression, and the eIF4B Antibody is used to control for loading and siRNA specificity.

Fluorescent Detection

Fluorescent Detection

Fluorescent detection of SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 in living HeLa cells 24 hours post-transfection, demonstrating nearly 100% transfection efficiency.

Directions for Use

CST recommends transfection with 100 nM mTOR siRNA 48 hours prior to cell lysis. See protocol on datasheet for transfection procedure.

Background

The mammalian target of rapamycin (mTOR, FRAP, RAFT) is a Ser/Thr protein kinase (1-3) that functions as an ATP and amino acid sensor to balance nutrient availability and cell growth (4,5). When sufficient nutrients are available, mTOR responds to a phosphatidic acid-mediated signal to transmit a positive signal to p70 S6 kinase and participate in the inactivation of the eIF4E inhibitor, 4E-BP1 (6). These events result in the translation of specific mRNA subpopulations. mTOR is phosphorylated at Ser2448 via the PI3 kinase/Akt signaling pathway and autophosphorylated at Ser2481 (7,8). mTOR plays a key role in cell growth and homeostasis and may be abnormally regulated in tumors. For these reasons, mTOR is currently under investigation as a potential target for anti-cancer therapy (9).

siRNA has been used to silence mTOR expression in 293-HEK cells causing decreased raptor expression, decreased p70 S6 kinase phosphorylation, and reduced cell size (9).

  1. Sabers, C.J. et al. (1995) J. Biol. Chem. 270, 815-822.
  2. Brown, E.J. et al. (1994) Nature 369, 756-758.
  3. Sabatini, D.M. et al. (1994) Cell 78, 35-43.
  4. Gingras, A.C. et al. (2001) Genes Dev. 15, 807-826.
  5. Dennis, P.B. et al. (2001) Science 294, 1102-1105.
  6. Fang, Y. et al. (2001) Science 294, 1942-1945.
  7. Nave, B.T. et al. (1999) Biochem. J. 344 Pt 2, 427-431.
  8. Peterson, R.T. et al. (2000) J. Biol. Chem. 275, 7416-7423.
  9. Huang, S. and Houghton, P.J. (2003) Curr. Opin. Pharmacol. 3, 371-377.
  10. Kim, D. H. et al. (2002) Cell 110, 163-175.

Application References

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Companion Products

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.

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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