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99732
GATOR Complex Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

GATOR Complex Antibody Sampler Kit #99732

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GATOR Complex Antibody Sampler Kit: Image 1

Immunoprecipitation of Mios from MCF7 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Mios (D12C6) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Mios (D12C6) Rabbit mAb.

GATOR Complex Antibody Sampler Kit: Image 2

Western blot analysis of extracts from various cell lines using NPRL2 (D8K3X) Rabbit mAb.

GATOR Complex Antibody Sampler Kit: Image 3

Immunoprecipitation of WDR59 from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is WDR59 (D4Z7A) Rabbit mAb. Western blot analysis was performed using WDR59 (D4Z7A) Rabbit mAb.

GATOR Complex Antibody Sampler Kit: Image 4

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

GATOR Complex 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).

GATOR Complex 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.

GATOR Complex Antibody Sampler Kit: Image 7

Western blot analysis of extracts from various cell lines using Mios (D12C6) Rabbit mAb.

GATOR Complex Antibody Sampler Kit: Image 8

Western blot analysis of extracts from various cell lines using WDR59 (D4Z7A) Rabbit mAb.

GATOR Complex Antibody Sampler Kit: Image 9

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).

GATOR Complex 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.

GATOR Complex Antibody Sampler Kit: Image 11

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

GATOR Complex Antibody Sampler Kit: Image 12

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).

GATOR Complex Antibody Sampler Kit: Image 13

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

GATOR Complex Antibody Sampler Kit: Image 14

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

GATOR Complex Antibody Sampler Kit: Image 15

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.

To Purchase # 99732T
Product # Size Price
99732T
1 Kit  (5 x 20 µl) $ 391

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Mios (D12C6) Rabbit mAb 13557 20 µl
  • WB
  • IP
H M R 98 Rabbit IgG
NPRL2 (D8K3X) Rabbit mAb 37344 20 µl
  • WB
  • IP
H Mk M R 41 Rabbit IgG
WDR59 (D4Z7A) Rabbit mAb 53385 20 µl
  • WB
  • IP
H Mk 110 Rabbit IgG
mTOR (7C10) Rabbit mAb 2983 20 µl
  • WB
  • IHC
  • IF
  • F
H Mk M R 289 Rabbit IgG
Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb 5536 20 µl
  • WB
  • IP
  • IF
H Mk M R 289 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The GATOR Complex Antibody Sampler Kit provides an economical means of detecting select components of the GATOR complex, mTOR and phospho-mTOR (Ser2448). The kit contains enough primary antibodies to perform at least two western blot experiments per antibody.

Specificity / Sensitivity

Mios (D12C6) Rabbit mAb recognizes endogenous levels of total Mios protein. NPRL2 (D8K3X) Rabbit mAb recognizes endogenous levels of total NPRL2 protein. WDR59 (D4Z7A) Rabbit mAb recognizes endogenous levels of total WDR59 protein. mTOR (7C10) Rabbit mAb detects endogenous levels of total mTOR protein. Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb detects endogenous levels of mTOR protein only when phosphorylated at Ser2448.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu730 of human Mios protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala211 of human NPRL2 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding His356 of human WDR59 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser2481 of human mTOR. Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser2448 of human mTOR protein.

Background

The mTORC1 kinase complex plays a critical role in cell growth regulation (1,2). mTORC1 activity is modulated by energy levels, growth factors, and amino acids (3,4). Four related GTPases (RagA, RagB, RagC, and RagD) interact with raptor in mTORC1, which is necessary and sufficient for mTORC1 activation in response to amino acid signals (1,2). The GAP Activity Towards Rags (GATOR) complex interacts with Rag GTPases and is made up of a pair of protein subcomplexes (5). The GATOR1 subcomplex includes the proteins DEPDC5, Nprl2 and Nprl3, and is a RagA and RagB GTPase-activating protein (GAP) that negatively regulates mTORC1 signaling. Conversely, the GATOR2 subcomplex (including Mios, WDR24, WDR59, Seh1L and Sec13 proteins) is a positive regulator of mTORC1 signaling (5).

The mammalian target of rapamycin (mTOR, FRAP, RAFT) is a Ser/Thr protein kinase (6-8) that functions as an ATP and amino acid sensor to balance nutrient availability and cell growth (9,10). 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 (11). 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 (12,13).

  1. Sancak, Y. et al. (2008) Science 320, 1496-501.
  2. Kim, E. et al. (2008) Nat Cell Biol 10, 935-45.
  3. Hay, N. and Sonenberg, N. (2004) Genes Dev 18, 1926-45.
  4. Wullschleger, S. et al. (2006) Cell 124, 471-84.
  5. Bar-Peled, L. et al. (2013) Science 340, 1100-6.
  6. Sabers, C.J. et al. (1995) J Biol Chem 270, 815-22.
  7. Brown, E.J. et al. (1994) Nature 369, 756-8.
  8. Sabatini, D.M. et al. (1994) Cell 78, 35-43.
  9. Gingras, A.C. et al. (2001) Genes Dev 15, 807-26.
  10. Dennis, P.B. et al. (2001) Science 294, 1102-5.
  11. Fang, Y. et al. (2001) Science 294, 1942-5.
  12. Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
  13. Peterson, R.T. et al. (2000) J Biol Chem 275, 7416-23.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.