Cell Signaling Technology

Product Pathways - Translational Control

mTOR Regulation Antibody Sampler Kit #9864

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
mTOR (7C10) Rabbit mAb #2983 40 µl W IHC-P IF-IC F H M R Mk (Hr) 289 Rabbit IgG
Phospho-Raptor (Ser792) Antibody #2083 40 µl W H M R 150 Rabbit
Phospho-PRAS40 (Thr246) (C77D7) Rabbit mAb #2997 40 µl W IP IHC-P H M R Mk 40 Rabbit IgG
PRAS40 (D23C7) XP® Rabbit mAb #2691 40 µl W IP IHC-P H M R Mk 40 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) (C) (Pg) (Hr) 289 Rabbit IgG
RagC (D31G9) XP® Rabbit mAb #5466 40 µl W IP IHC-P H M R Mk 50 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  C=Chicken  Pg=Pig  Hr=Horse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Each antibody in the mTOR Regulation Antibody Sampler Kit detects endogenous levels of its target protein. Activation state antibodies detect only target proteins phosphorylated at indicated residues. Phospho-Raptor (Ser792) Antibody may also detect non-specific signals of various molecular weights.

Western Blotting

Western Blotting

Western blot analysis of C2C12 or 293 cells, untreated or treated with AICAR (0.5 mM for 30 minutes) or oligomycin (0.5 μM for 30 minutes), using Phospho-Raptor (Ser792) Antibody #2083 (upper and lower left) or Raptor Antibody #2280 (upper and lower right). *Cross-reacting bands at 200 kDa.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell types using PRAS40 (D23C7) XP® Rabbit mAb #2691.

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 serum starved H3255, Mkn45 and NIH/3T3 cells, untreated or treated with either Iressa (1 μM, 3 hours), Su11274 (1 μM, 3 hours) or insulin (150 nM, 15 minutes), using Phospho-PRAS40 (Thr246) (C77D7) Rabbit mAb #2997 (upper) or PRAS40 (D23C7) Rabbit mAb #2691 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using RagC (D31G9) XP® Rabbit mAb #5466.

Western Blotting

Western Blotting

Western blot analysis of extracts from serum-starved NIH/3T3 cells, untreated or insulin-treated (150 nM, 5 minutes), alone or in combination with λ-phosphatase, using Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb #5536 (upper) or mTOR (7C10) Rabbit mAb #2983.


Description

The mTOR Regulation Sampler Kit provides an economical means to evaluate the regulation of mTOR signaling by such proteins as phosphorylated Raptor, RagC and PRAS40. The kit contains enough primary and secondary antibodies to perform four Western blot experiments per primary antibody.

Source / Purification

Phospho-specific polyclonal antibody is produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser792 of human raptor. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Phospho-specific monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to the sequence surrounding Thr246 of human PRAS40 and Ser2448 of human mTOR. Total protein monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Ser2481 of human mTOR, the sequence of human PRAS40, and the residues near the aminoterminus of human RagC.

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) was identified as an mTOR binding partner that mediates mTOR signaling to downstream targets (10,11). Raptor binds to mTOR substrates, including 4E-BP1 and p70 S6 kinase, through their TOR signaling (TOS) motifs and is required for mTOR-mediated phosphorylation of these substrates (12,13). PRAS40 interacts with raptor in insulin-deprived cells and inhibits the activation of the mTORC1 pathway. Phosphorylation of PRAS40 by Akt at Thr246 relieves PRAS40 inhibition of mTORC1 (14). Recently raptor has been identified as a direct substrate of the AMP-activated protein kinase (AMPK) (15). AMPK phosphorylates raptor on Ser722/Ser792 (15). This phosphorylation is essential for inhibition of the raptor-containing mTOR complex 1 (mTORC1) and induces cell cycle arrest when cells are stressed for energy (15). These findings suggest that raptor is a critical switch that correlates cell cycle progression with energy status. The activity of mTORC1 kinase complex is modulated by energy levels, growth factors and amino acids (16,17). Recent studies found that RagA, RagB, RagC and RagD, the four related GTPases, interact with raptor in the mTORC1 complex (18,19). These interactions are both necessary and sufficient for mTORC1 activation in response to amino acid signals (18,19).

  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, 177-89.
  11. Kim, D.H. et al. (2002) Cell 110, 163-75.
  12. Beugnet, A. et al. (2003) J Biol Chem 278, 40717-22.
  13. Nojima, H. et al. (2003) J Biol Chem 278, 15461-4.
  14. Vander Haar, E. et al. (2007) Nat Cell Biol 9, 316-23.
  15. Gwinn, D.M. et al. (2008) Mol Cell 30, 214-26.
  16. Hay, N. and Sonenberg, N. (2004) Genes Dev 18, 1926-45.
  17. Wullschleger, S. et al. (2006) Cell 124, 471-84.
  18. Sancak, Y. et al. (2008) Science 320, 1496-501.
  19. Kim, E. et al. (2008) Nat Cell Biol 10, 935-45.

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