Cell Signaling Technology

Product Pathways - Translational Control

mTOR Substrates Antibody Sampler Kit #9862

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-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb #9234 40 µl W H M R Mk (C) 70, 85 Rabbit IgG
Phospho-p70 S6 Kinase (Ser371) Antibody #9208 40 µl W H M R Mk 70, 85 Rabbit
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855 40 µl W IHC-P IF-IC F H M R Mk Dm 15 to 20 Rabbit IgG
Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb #5536 40 µl W IP IF-IC H M Mk (R) (C) (Pg) (Hr) 289 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

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  Dm=D. melanogaster  Pg=Pig  Hr=Horse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Each antibody in the mTOR Substrates Antibody Sampler Kit detects endogenous levels of its target protein. While activation state antibodies typically detect only target proteins phosphorylated at indicated residues, some cross-reaction can occur with related proteins phosphorylated at analogous sites.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells using 4E-BP1 Antibody #9452 (upper) and Phospho-4E-BP1 (Thr37/46) Antibody #2855 (lower). The cells were starved for 24 hours in serum-free medium and underwent a 1 hour amino acid deprivation. Amino acids were replenished for 1 hour. Cells were then either untreated (-) or treated with 100 nM insulin (+) for 30 minutes.

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 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.


Western Blotting

Western Blotting

Western blot analysis of lysates from 293 cells grown in low serum, then treated with 20% serum for 30 minutes alone or after 1 hour preincubation with rapamycin (10 nM) #9904 or LY294002 (50 uM) #9901, using Phospho-p70 S6 Kinase (Ser371) Antibody #9208 (upper) or p70 S6 Kinase Antibody #9202 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from serum starved or serum treated (20%) 293, NIH/3T3, and PC12 cells, using Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb #9234 (upper) or p70 S6 Kinase (49D7) Rabbit mAb #2708 (lower).

Description

The mTOR Substrates Antibody Sampler Kit provides an economical means to evaluate the signaling of mTOR to downstream substrates including p70 S6 Kinase and 4E-BP1. The kit contains enough primary and secondary antibodies to perform four Western blot experiments per primary antibody.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser371 of human p70 S6 kinase. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Phospho-specific rabbit monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Thr389 of human p70 S6 kinase, Thr37 and Thr46 of mouse 4E-BP1 and the Ser2448 site of human mTOR. The mTOR (7C10) Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser2481 of human mTOR.

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 are 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, 177-89.
  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. Oshiro, N. et al. (2004) Genes Cells 9, 359-66.
  15. Kim, D.H. et al. (2003) Mol Cell 11, 895-904.
  16. Sarbassov, D.D. et al. (2004) Curr Biol 14, 1296-302.
  17. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.

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