Cell Signaling Technology

Product Pathways - Protein Translation

mTOR Substrates Antibody Sampler Kit #9862

4E-BP1   4ebp   4EBP1   BP1   mTOR   mTORC   p70   p70 S6   p70 S6 kinase   p70s6   p70S6K   S6   TOR  

REACTIVITY

No. Size Price
9862T 1 Kit ( 5 x 20 µl ) ¥4,653.00 现货查询 购买询价
Kit Includes Quantity Applications Reactivity Homology† MW (kDa) Isotype
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855 20 µl W,IHC-P,IF-IC,F, H,M,R,Mk,Dm, 15 to 20 Rabbit IgG
mTOR (7C10) Rabbit mAb #2983 20 µl W,IHC-P,IF-IC,F, H,M,R,Mk, Hr, 289 Rabbit
Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb #5536 20 µl W,IP,IF-IC, H,M,R,Mk, R,C,Pg,Hr, 289 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl W, Goat
Phospho-p70 S6 Kinase (Ser371) Antibody #9208 20 µl W, H,M,R,Mk, 70, 85 Rabbit
Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb #9234 20 µl W, H,M,R,Mk, C, 70, 85 Rabbit IgG

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.

mTOR Substrates Antibody Sampler Kit的每个抗体检测内源性水平的目标蛋白。虽然激活状态的抗体通常只能检测到特定残基磷酸化的靶点蛋白,但当相关蛋白在同源位点存在磷酸化修饰时可能发生交叉反应。

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.

多克隆抗体是由合成的针对人源的针对人p70 S6 激酶Ser371位点磷酸化肽段免疫动物生产的, 并用蛋白质A和肽亲和层析技术纯化。磷酸化特异性兔单克隆抗体是由合成的针对人源的p70 S6 激酶Thr389位点,鼠源的针对4E-BP1 蛋白的Thr37和Thr46位点和人源的针对mTOR蛋白Ser2448位点磷酸化肽段免疫动物生产的。

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.

mTOR Substrates Antibody Sampler Kit为您提供一种检测mTOR信号到下游底物如p70 S6 激酶和 4E-BP1 蛋白的信号通路的经济方式。Kit包含足够的可以做四次Western blot实验的一抗和二抗。

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

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).

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

Flow Cytometry

Flow Cytometry

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

IHC-P (paraffin)

IHC-P (paraffin)

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

IHC-P (paraffin)

IHC-P (paraffin)

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).

Western Blotting

Western Blotting

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

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

IHC-P (paraffin)

IHC-P (paraffin)

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

IHC-P (paraffin)

IHC-P (paraffin)

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)).

Flow Cytometry

Flow Cytometry

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).

IHC-P (paraffin)

IHC-P (paraffin)

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

IHC-P (paraffin)

IHC-P (paraffin)

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).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells treated with LY294002 (left) or 20% serum (right) and labeled with Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

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 blot分析不同细胞提取物,所用抗体为mTOR (7C10) Rabbit mAb兔单抗 #2983

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).

Western blot 分析血清饥饿和20%血清处理的293, NIH/3T3, 和 PC12 细胞提取物,所用抗体为Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb兔单抗 #9234 (上) 或 p70 S6 Kinase (49D7) Rabbit mAb 兔单抗#2708 (下)。

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 blot分析生长在低浓度血清中的293细胞,然后用20%的血清单独处理30分钟,或与雷帕霉素(10 nM) #9904 or LY294002 (50 uM) #9901预孵育1小时,所用抗体为Phospho-p70 S6 Kinase (Ser371) Antibody #9208 (上) 或 p70 S6 Kinase Antibody #9202 (下)

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 blot分析293T细胞提取物,所用抗体为4E-BP1 Antibody #9452 (u上) 和 Phospho-4E-BP1 (Thr37/46) Antibody #2855 (下). 细胞在无血清的培养液中饥饿24小时后,去除氨基酸1小时。在加入氨基酸1小时。未处理或采用100 nM insulin (+) 处理30分钟。

Western Blotting

Western Blotting

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.

IF-IC

IF-IC

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).

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

IF-IC

IF-IC

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).

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 blot分析血清饥饿的NIH/3T3细胞,未处理组和胰岛素处理组 (150 nM, 5 分钟), 单独或与λ-磷酸酶联合组,所用抗体为Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb兔单抗 #5536 (上) 或 mTOR (7C10) Rabbit mAb兔单抗 #2983

IHC-P (paraffin)

IHC-P (paraffin)

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

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).

哺乳动物雷帕霉素(rapamycin)靶蛋白(mTOR,FRAP,RAFT)是一种丝氨酸/苏氨酸蛋白激酶(1-3),其作为ATP和氨基酸的传感器,可以有效调控营养供应及细胞的生长(4,5)。当营养充分时,mTOR响应磷脂酸介导的信号,进而正向调节p70 S6 激酶,并参与失活eIF4E的抑制剂4E-BP1。这些信号导致特定的mRNA亚群的翻译。mTOR可以被Akt信号通路的PI3K激酶磷酸化修饰Ser2448位点,也能自我磷酸化Ser2481位点(7,8)。mTOR的在细胞生长和动态平衡中起着关键作用,在肿瘤中可能存在调控失常。因此,目前很多研究将mTOR作为一个潜在的抗肿瘤治疗靶点(9)。

mTOR的调节相关蛋白(Raptor)与mTOR互作调节mTOR信号通路下游靶点(10,11)。Raptor与4E-BP1和p70 S6激酶等mTOR底物的TOR信号(TOS)结构域结合,协助mTOR调控底物磷酸化(12,13)。FKBP12-rapamycin(雷帕霉素)复合物与mTOR结合可以抑制mTOR-Raptor互作,这可能是雷帕霉素(rapamycin)抑制mTOR活性的一种机制(14)。mTOR-Raptor的相互作用及营养物质和(或)雷帕霉素(rapamycin)对其的调节都依赖于一种叫GβL的蛋白(15)。作为位于mTOR和rictor(rapamycin-insensitive companion of mTOR)之间、雷帕霉素非敏感复合物的组成成分,GβL可能介导rictor-mTOR到PKCα及其他下游靶点的信号转导(16)。rictor-mTOR复合物调节Akt / PKB Ser473位点的磷酸化(曾经被认为是被PDK2所磷酸化),进而促使PDK1磷酸化Akt / PKB的Ser308位点,最终充分激活Akt / PKB(17)。

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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