Interested in promotions? | Click here >>
9862
mTOR Substrates Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

mTOR Substrates Antibody Sampler Kit #9862

Reviews ()
Citations (0)
mTOR Substrates Antibody Sampler Kit: Image 1

Flow cytometric analysis of 293 cells using mTOR (7C10) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).

mTOR Substrates Antibody Sampler Kit: Image 2

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 (upper), or p70 S6 Kinase (49D7) rabbit mAb #2708 (lower).

mTOR Substrates Antibody Sampler Kit: Image 3

Western blot analysis of lysates from unsynchronized (U) and nocodazole (N) treated (50ng/ml for 48 hours) HT29 cells using Phospho-p70 S6 Kinase (Ser371) Antibody (B) and p70 S6 Kinase Antibody #9202 (D). Incubation of the nitrocellulose membrane with calf intestinal alkaline phosphatase (CIP) after Western transfer abolishes the phospho-p70 S6 Kinase signal (A), but has no effect on the total p70 S6 kinase signal (C).

mTOR Substrates Antibody Sampler Kit: Image 4

Flow cytometric analysis of Jurkat cells, untreated (green) or LY294002, Wortmannin and U0126-treated (blue), using Phospho-4E-BP1 (Thr36/46) (236B4) Rabbit mAb compared to a nonspecific negative control antibody (red).

mTOR Substrates Antibody Sampler Kit: Image 5

Confocal immunofluorescent analysis of HeLa cells, rapamycin-treated (#9904, 10 nM for 2 hours, left), insulin-treated (150 nM for 6 minutes, middle) or insulin- and λ-phosphatase-treated (right), using Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

mTOR Substrates Antibody Sampler Kit: Image 6

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

mTOR Substrates Antibody Sampler Kit: Image 7

Confocal immunofluorescent analysis of mouse embryonic fibroblast (MEF) cells using mTOR (7C10) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

mTOR Substrates Antibody Sampler Kit: Image 8

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 (10nM) #9904 or LY294002 (50uM) #9901, using Phospho-p70 S6 Kinase (Ser371) Antibody (upper) or p70 S6 Kinase Antibody #9202 (lower).

mTOR Substrates Antibody Sampler Kit: Image 9

Confocal immunofluorescent analysis of 293 cells, expressing either non-targeting shRNA (top) or shRNA targeting 4E-BP1/2 (bottom), using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (green). To confirm phospho-specificity, cells were treated with an inhibitor cocktail consisting of LY294002 #9901, U0126 #9903, and Rapamycin #9904 (50 μM; 10 μm; 10 nM; 2 hr) (left), stimulated with insulin (100 nM, 30 min; middle), or processed with λ-phosphatase following insulin stimulation (right). Red = Propidium Iodide (PI)/RNase Staining Solution (#4087).

mTOR Substrates Antibody Sampler Kit: Image 10

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 (upper) or mTOR (7C10) Rabbit mAb #2983.

mTOR Substrates Antibody Sampler Kit: Image 11

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing cytoplasmic localization using mTOR (7C10) Rabbit mAb.

mTOR Substrates Antibody Sampler Kit: Image 12

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb.

mTOR Substrates Antibody Sampler Kit: Image 13

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using mTOR (7C10) Rabbit mAb in the presence of control peptide (left) or mTOR Blocking Peptide #1072 (right).

mTOR Substrates Antibody Sampler Kit: Image 14

Immunohistochemical analysis of paraffin-embedded human lymphoma using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb.

mTOR Substrates Antibody Sampler Kit: Image 15

Immunohistochemical analysis of paraffin-embedded mouse brain using mTOR (7C10) Rabbit mAb.

mTOR Substrates Antibody Sampler Kit: Image 16

Immunohistochemical analysis using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb on SignalSlide (TM) Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells untreated (left) or LY294002-treated (right)).

mTOR Substrates Antibody Sampler Kit: Image 17

Western blot analysis of extracts from 293, A431, COS, C6, and C2C12 cells, using mTOR (7C10) Rabbit mAb.

mTOR Substrates Antibody Sampler Kit: Image 18

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb in the presence of control peptide (left) or Phospho-4E-BP1 (Thr37/46) Blocking Peptide #1052 (right).

mTOR Substrates Antibody Sampler Kit: Image 19

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® mTOR siRNA II (+), using mTOR (7C10) Rabbit mAb #2983 and α-Tubulin (11H10) Rabbit mAb #2125. mTOR (7C10) Rabbit mAb confirms silencing of mTOR expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of mTOR siRNA.

mTOR Substrates Antibody Sampler Kit: Image 20

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.

To Purchase # 9862T
Product # Size Price
9862T
1 Kit  (5 x 20 µl) $ 391

Our US Office is Closed

Our US office is closed in observance of Thanksgiving. We will reopen on Monday, November 30th.

Thank you for your patience.

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
mTOR (7C10) Rabbit mAb 2983 20 µl
  • WB
  • IHC
  • IF
  • F
H M R Mk 289 Rabbit IgG
Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb 9234 20 µl
  • WB
H M R Mk 70, 85 Rabbit IgG
Phospho-p70 S6 Kinase (Ser371) Antibody 9208 20 µl
  • WB
H M R Mk 70, 85 Rabbit 
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb 2855 20 µl
  • WB
  • IHC
  • IF
  • F
H M R Mk Dm 15 to 20 Rabbit IgG
Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb 5536 20 µl
  • WB
  • IP
  • IF
H M R Mk 289 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product 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 two Western blot experiments per primary antibody.

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.

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. et al. (2004) Curr. Biol. 14, 1296-302.
  17. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.