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9864
mTOR Regulation Antibody Sampler Kit

mTOR Regulation Antibody Sampler Kit #9864

Western Blotting Image 1

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

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Western Blotting Image 2

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.

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Western Blotting Image 3

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 (upper and lower left ) or Raptor Antibody #2280 (upper and lower right).

*Cross-reacting bands at 200 kDa.

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Western Blotting Image 4

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

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Western Blotting Image 5

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

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Western Blotting Image 6

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

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Western Blotting Image 7

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.

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Western Blotting Image 8

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.

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IF-IC Image 9

Confocal immunofluorescent analysis of HeLa cells, rapamycin-treated (#9904, 10 μM 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).

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Western Blotting Image 10

Western blot analysis of wild-type (WT) and AMPKα1 and α2 knockout (KO) mouse embryonic fibroblasts (MEFs), untreated or treated with AICAR (2 mM for 1 hour), using Phospho-Raptor (Ser792) Antibody (upper) or Raptor Antibody #4978 (lower). (Image provided by Dr. Reuben Shaw, Salk Institute for Biological Studies).

*Cross-reacting bands at 60, 70 and 240 kDa

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IHC-P (paraffin) Image 11

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using PRAS40 (D23C7) XP® Rabbit mAb.

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Western Blotting Image 12

Western blot analysis of extracts from serum starved HeLa cells, untreated or treated with insulin (100 nM, 5 minutes) or with insulin and λ phosphatase, using Phospho-PRAS40 (Thr246) (C77D7) Rabbit mAb (upper) or PRAS40 Antibody #2610 (lower).

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IHC-P (paraffin) Image 13

Immunohistochemical analysis of paraffin-embedded mouse colon using RagC (D31G9) XP® Rabbit mAb.

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IHC-P (paraffin) Image 14

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

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IHC-P (paraffin) Image 15

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-PRAS40 (Thr246) (C77D7) Rabbit mAb in the presence of control peptide (left) or antigen specific peptide (right).

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IHC-P (paraffin) Image 16

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using RagC (D31G9) XP® Rabbit mAb.

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IHC-P (paraffin) Image 17

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).

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IHC-P (paraffin) Image 18

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-PRAS40 (Thr246) (C77D7) Rabbit mAb.

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IHC-P (paraffin) Image 19

Immunohistochemical analysis of paraffin-embedded human lymphoma using RagC (D31G9) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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IHC-P (paraffin) Image 20

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

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IHC-P (paraffin) Image 21

Immunohistochemical analysis of paraffin-embedded metastatic SKOV-3 tumor in mouse lung using Phospho-PRAS40 (Thr246) (C77D7) Rabbit mAb.

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Flow Cytometry Image 22

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

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IF-IC Image 23

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).

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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-mTOR (Ser2448) (D9C2) XP® Rabbit mAb 5536 20 µl
  • WB
  • IP
  • IF
H M R Mk 289 Rabbit IgG
Phospho-Raptor (Ser792) Antibody 2083 20 µl
  • WB
H M R 150 Rabbit 
PRAS40 (D23C7) XP® Rabbit mAb 2691 20 µl
  • WB
  • IP
  • IHC
H M R Mk 40 Rabbit IgG
Phospho-PRAS40 (Thr246) (C77D7) Rabbit mAb 2997 20 µl
  • WB
  • IP
  • IHC
H M R Mk 40 Rabbit IgG
RagC (D31G9) XP® Rabbit mAb 5466 20 µl
  • WB
  • IP
  • IHC
H M R Mk 50 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

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 two Western blot experiments per primary antibody.

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.

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.

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. Sancak, Y. et al. (2008) Science 320, 1496-501.
  11. Kim, E. et al. (2008) Nat Cell Biol 10, 935-45.
  12. Vander Haar, E. et al. (2007) Nat Cell Biol 9, 316-23.
  13. Hay, N. and Sonenberg, N. (2004) Genes Dev 18, 1926-45.
  14. Gwinn, D.M. et al. (2008) Mol Cell 30, 214-26.
  15. Wullschleger, S. et al. (2006) Cell 124, 471-84.
  16. Hara, K. et al. (2002) Cell 110, 177-189.
  17. Kim, D.H. et al. (2002) Cell 110, 163-175.
  18. Beugnet, A. et al. (2003) J. Biol. Chem. 278, 40717-40722.
  19. Nojima, H. et al. (2003) J. Biol. Chem. 278, 15461-15464.
Entrez-Gene Id
2475 , 84335 , 64121 , 57521
Swiss-Prot Acc.
P42345 , Q96B36 , Q9HB90 , Q8N122
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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