Cell Signaling Technology

Product Pathways - Translational Control

mTOR Pathway Antibody Sampler Kit #9964

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-mTOR (Ser2481) Antibody #2974 40 µl W H M R Mk 289 Rabbit
mTOR (7C10) Rabbit mAb #2983 40 µl W IHC-P IF-IC F H M R Mk 289 Rabbit IgG
Raptor (24C12) Rabbit mAb #2280 40 µl W IP H M R Mk 150 Rabbit IgG
Rictor (53A2) Rabbit mAb #2114 40 µl W H M R Mk 200 Rabbit IgG
GβL (86B8) Rabbit mAb #3274 40 µl W IP H M R Mk 37 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat
Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb #5536 40 µl W IP IF-IC H M Mk (R) 289 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Each total antibody in the mTOR Pathway Sampler Kit recognizes only its specific target. Each phospho-specific antibody detects the intended target only when phosphorylated at the indicated site.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell types using Rictor (53A2) Rabbit mAb #2114.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell types using Raptor (24C12) Rabbit mAb #2280.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293 cells (starved for 30 hours), untreated or 20% FBS-treated for 30 minutes, using Phospho-mTOR (Ser2481) Antibody #2974.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell types using mTOR (7C10) Rabbit mAb #2983.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines, using GβL (86B8) Rabbit mAb #3274.

Description

The mTOR Pathway Antibody Sampler Kit contains reagents to investigate the control of protein translation, cell growth, and proliferation through mTOR signaling within cells. The kit contains enough primary and secondary antibodies to perform four Western blot experiments per primary antibody.

Source / Purification

Polyclonal antibody is produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser2481 of human mTOR. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser2448 of human mTOR, Gln1681 of human Rictor, Gln210 of human GβL and human Raptor.

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).

The regulatory associated protein of mTOR (Raptor) interacts with mTOR to mediate mTOR signaling to downstream targets (10,11). Raptor binds to mTOR substrates, such as 4E-BP1 and p70 S6 kinase, through their TOR signaling (TOS) motifs and is required for mTOR-mediated substrate phosphorylation (12,13). Binding of the FKBP12-rapamycin complex to mTOR inhibits mTOR-raptor interaction, which suggests a mechanism for the inhibition of mTOR signaling by rapamycin (14). This mTOR-raptor interaction and its regulation by nutrients and/or rapamycin is dependent on a protein called GβL (15). GβL is part of the rapamycin-insensitive complex between mTOR and rictor (rapamycin-insensitive companion of mTOR) and may mediate rictor-mTOR signaling to PKCα and other downstream targets (16). The rictor-mTOR complex has been identified as the previously elusive PDK2 responsible for the phosphorylation of Akt/PKB at Ser473, which is required for PDK1 phosphorylation of Akt/PKB at Thr308 and full activation of Akt/PKB (17).

  1. Sabers, C.J. et al. (1995) J Biol Chem 270, 815-22.
  2. Brown, E.J. et al. (1994) Nature 369, 756-8.
  3. Sabatini, D.M. et al. (1994) Cell 78, 35-43.
  4. Gingras, A.C. et al. (2001) Genes Dev 15, 807-26.
  5. Dennis, P.B. et al. (2001) Science 294, 1102-5.
  6. Fang, Y. et al. (2001) Science 294, 1942-5.
  7. Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
  8. Peterson, R.T. et al. (2000) J Biol Chem 275, 7416-23.
  9. Huang, S. and Houghton, P.J. (2003) Curr Opin Pharmacol 3, 371-7.
  10. Hara, K. et al. (2002) Cell 110, 177-189.
  11. Kim, D.H. et al. (2002) Cell 110, 163-175.
  12. Beugnet, A. et al. (2003) J. Biol. Chem. 278, 40717-40722.
  13. Nojima, H. et al. (2003) J. Biol. Chem. 278, 15461-15464.
  14. Oshiro, N. et al. (2004) Genes Cells 9, 359-366.
  15. Kim, D.H. et al. (2003) Mol. Cell 11, 895-904.
  16. Sarbassov, D.D. et al. (2004) Curr. Biol. 14, 1296-1302.
  17. Sarbassov, D.D. et al. (2005) Science 307, 1098-1101.

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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.

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