Cell Signaling Technology

Product Pathways - Translational Control

Translational Control Sampler Kit #9918

Kit Includes Quantity Applications Reactivity MW (kDa) Source
Phospho-Mnk1 (Thr197/202) Antibody # 2111 40 microliters W H M R Mk 50 Rabbit
Phospho-S6 Ribosomal Protein (Ser235/236) Antibody # 2211 40 microliters W IP IHC-P IHC-F IF-IC F H M R Mk (C) (X) 32 Rabbit
Phospho-Akt (Ser473) Antibody # 9271 40 microliters W IP IF-IC F H M R Pg C Hm Dr Dg (B) 60 Rabbit
Phospho-eIF4E (Ser209) Antibody # 9741 40 microliters W H M R Mk 25 Rabbit
Phospho-4E-BP1 (Ser65) Antibody # 9451 40 microliters W IP H M R Mk 15 to 20 Rabbit
Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb # 9234 40 microliters W H M R 70, 85 Rabbit
LY294002 (PI3 Kinase Inhibitor) # 9901 0.3 milligrams 307
Rapamycin (FRAP/mTOR Inhibitor) # 9904 2 micrograms 914.2
Anti-rabbit IgG, HRP-linked Antibody # 7074 100 microliters Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IHC-F=Immunohistochemistry (Frozen)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Pg=Pig  C=Chicken  Hm=Hamster  B=Bovine  Dr=Drosophila  X=Xenopus  Dg=Dog

Specificity / Sensitivity

Each phospho-specific antibody detects the intended target only when phosphorylated at the indicated site.

Western Blotting

Western Blotting

Western blot analysis of extracts from FBS-treated NIH/3T3 cells using Phospho-Mnk1 (Thr197/202) Antibody #2111.

Western Blotting

Western Blotting

Western blot analysis of FLAG immunoprecipitates from CHO cells transfected with Mnk1-FLAG, using Phospho-Mnk1 (Thr197/202) Antibody #2111 (upper) and FLAG antibody (lower). (Mnk1-FLAG plasmid provided by Dr. Nahum Sonenberg, McGill University, Montreal, Quebec.)

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 Blotting

Western Blotting

Western blot analysis of extracts from PDGF treated NIH/3T3 cells, using Phospho-Akt (Ser473) Antibody #9271 (upper) or Akt Antibody #9272 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts 293 cells, untreated or treated with 20% FBS, using Phospho-4E-BP1 (Ser65) Antibody #9451 or 4E-BP1 antibody. (293 cells provided by Dr. Nahum Sonenberg, McGill University, Montreal, Quebec.)

Western Blotting

Western Blotting

Western blot analysis of extracts from C6 and MEF untreated cells using Phospho-4E-BP1 (Ser65) Antibody #9451 (upper) or 4E-BP1 (53H11) Rabbit mAb (lower).


Western Blotting

Western Blotting

Western blot analysis of extracts from NIH/3T3 cells, untreated or treated with serum, PD98059 or Dexamethasone, using Phospho-eIF4E (Ser209) Antibody #9741 (upper) or eIF4E Antibody #9742 (lower).

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical staining of paraffin-embedded human melanoma, showing cytoplasmic localization of phosphorylated S6 ribosomal protein, using Phospho-S6 Ribosomal Protein (Ser235/236) Antibody.

IF-IC

IF-IC

Immunofluorescent analysis of HeLa cells, untreated (A), 20% serum-treated (B) and serum-treated after rapamycin pretreatment (C), using Phospho-S6 Ribosomal Protein (Ser235/236) Antibody.


Source / Purification

Antibodies are produced by immunizing rabbits with synthetic phospho-peptides (KLH coupled) corresponding to residues surrounding Ser473 of mouse Akt; Ser209 of human elF4E; Ser65 of mouse 4E-BP1; and Thr389 of human p70 S6 kinase. Antibodies are purified by protein A and peptide affinity chromatography.

Background

Key steps in translational control occur at the level of eukaryotic initiation factor 4F (eIF4F) and p70 S6 kinase regulation. eIF4F is a complex whose functions include the recognition of the mRNA 5' cap structure. Several stimuli, such as insulin and various growth and survival factors, regulate the eIF4F complex and p70 S6 kinase primarily by triggering a signaling cascade dependent on sequential activation of PI3K, Akt/PKB and mTOR/FRAP kinases. Akt is activated by phosphorylation within the C-terminus at Ser473 and within the activation loop at Thr308 by phospholipid-dependent kinases. Inactivation in vivo of PI3K by the highly selective inhibitor LY294002 inhibits Akt and downstream elements of this cascade. Direct phosphorylation of mTOR/FRAP at Ser2448 by Akt is a key regulatory event controlling its kinase activity. mTOR/FRAP activity can be effectively blocked by Rapamycin, leading to inactivation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1), an inhibitor of translation initiation, and activation of p70 S6 kinases. Inactivation of 4E-BP1 by sequential phosphorylation causes the release of eIF4E, which, together with eIF4G and other factors, forms a functional eIF4F cap binding complex. p70 S6 kinases phosphorylates the 40S ribosomal subunit protein S6 and stimulates the translation of 5' oligopyrimidine tract containing mRNAs. The Erk pathway is also involved in regulation at this level by regulating the eIF4E kinase, Mnk1, and activating p70 S6 kinase. Tuberin, a product of the tumor supressor gene TSG2, is directly phosphorylated atThr1462 by Akt/PKB. Tuberin inhibits the mammalian target of rapamycin, mTOR, which results in inhibition of p70 S6 kinase and activation of 4E-BP1 and, therefore, inhibition of translation.

Description: The Translational Control Sampler Kit provides a fast and economical means of evaluating the activity of pathways regulating translation. The kit contains enough primary and secondary antibodies to perform four Western mini-blot experiments, as well as specific inhibitors of PI3 kinase and mTOR/FRAP.

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