Cell Signaling Technology

Product Pathways - Translational Control

Translational Control Antibody Sampler Kit #9918

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 40 µl W IP IHC-P IHC-F IF-IC F H M R Hm Mk Dm Z B (C) (X) (Dg) (Pg) 60 Rabbit IgG
Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb #9234 40 µl W H M R Mk (C) 70, 85 Rabbit IgG
Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb #4858 40 µl W IHC-P IHC-F IF-IC F H M R Mk Mi Sc (C) (Pg) 32 Rabbit IgG
Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb #3398 40 µl W IP IHC-P H M R Mk Dm 38 Rabbit IgG
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855 40 µl W IHC-P IF-IC F H M R Mk Dm 15 to 20 Rabbit IgG
Phospho-eIF4E (Ser209) Antibody #9741 40 µl W H M R Mk 25 Rabbit
Rapamycin #9904 2 micrograms 914.2
LY294002 #9901 0.3 milligrams 307.34
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl 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  Hm=Hamster  Mk=Monkey  C=Chicken  Mi=Mink  Dm=D. melanogaster  X=Xenopus  Z=Zebrafish  B=Bovine  Dg=Dog  Pg=Pig  Sc=S. cerevisiae
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Each phospho-specific antibody in the Translational Control Antibody Sampler Kit detects the intended target only when phosphorylated at the indicated site. Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb may cross-react with 4E-BP2 and 4E-BP3 when phosphorylated at equivalent sites.

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 C2C12 cells, untreated or thapsigargin-treated, using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb #3398 (upper) or eIF2α Antibody #9722 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from PC-3 cells, untreated or LY294002/wortmannin-treated, and NIH/3T3 cells, serum-starved or PDGF-treated, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).


Western Blotting

Western Blotting

Western blot analysis of extracts from PC12 and NIH/3T3 cells, treated with λ phosphatase, 20% FBS (20 min) or 100 ng/ml PDGF (20 min) as indicated, using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb #4858 (upper) or S6 Ribosomal Protein (5G10) Rabbit mAb #2217 (lower).

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


Description

The Translational Control Antibody Sampler Kit provides a fast and economical means of evaluating multiple proteins involved in translational control. The kit contains enough primary and secondary antibody to perform four Western blot experiments, as well as specific inhibitors of PI3 kinase and mTOR/FRAP.

Source / Purification

Phospho-specific polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser209 of human elF4E. 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 Ser473 of mouse Akt, Thr37 and Thr46 of mouse 4E-BP1, Ser51 of human eIF2α, Ser235 and Ser236 of human ribosomal protein S6, and Thr389 of human p70 S6 kinase.

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.

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Application References

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Protocols

* Product-specific protocol.

Companion Products

Selected rabbit monoclonal antibodies are produced under license (granting certain rights including those under U. S. Patent No. 5,675,063 and/orU.S.S.N. 11/476,277) from Epitomics, Inc.

For Research Use Only. Not For Use In Diagnostic Procedures.

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