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Primary Antibodies
4E-BP1 [pT46] Antibody
Background
Eukaryotic initiation factor 4E binding protein 1 (4E-BP1), also known as PHAS, is a ~20 kDa member of a family of eIF4E-binding proteins whose binding affinity to eIF4E is regulated by its phosphorylation. It inhibits cap-dependent translation by binding to eIF4E on the same site that overlaps the binding site for eIF4G, preventing its binding to the latter and eventually leading to an increase in mRNA translation. The phosphorylatin of 4E-BP1 is critical in determining cell fate by controlling translation initiation and apoptotic potency. 4E-BP1 is hyperphosphorylated in response to several external stimuli including hormones, growth factors, mitogens, cytokines and G-protein–coupled receptors and in response to stress conditions including nutrient deprivation. The phosphorylation of 4E-BP1 increases in response to activated phosphoinositol 3’-kinase (PI-3K) or its downstream effector Akt/PKB. 4E-BP1 is believed to mediate PI-3K and FRAP/mTOR signaling and is phosphorylated on at least six serine and threonine sites (Thr 37, Thr 46, Ser 65, Thr 70, Ser 83, and Ser 112). The phosphorylation of these sites is believed to occur in an orderly fashion where phosphorylation of threonine 37 and 46 by FRAP/mTOR is a priming step for subsequent phosphorylation of 4E-BP1 at the carboxy-terminal sites.
![4E-BP1 [pT46] Antibody](http://www.prosci-inc.com/shop/images/XBP-4031.gif)
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Description
Lysates prepared from NIH3T3 cells left untreated or treated with PI-3K inhibitor LY2904002 (lanes 2 & 3), prior to PDGF stimulation (lanes 1 & 3) and from EGF-treated HEK293 cells (4-8), were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were then left untreated (lanes 1-7) or treated with Lambda phosphatase (8), blocked with a 3% BSA-TBST buffer for one hour at room temperature, and incubated with 4E-BP1 [pT46] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (lanes 1 & 2), non-phosphopeptide corresponding to the immunogen (3), a generic phospho-threonine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal™ method.
The data show that the phosphorylation of 4E-BP1 on threonine 46 is blocked by PI-3K inhibitor and that phosphatase treatment eliminates the signal, thereby demonstrating the phospho-specificity of the antibody.
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Source
4E-BP1 antibody was produced against a chemically synthesized phosphopeptide derived from the region of human 4E-BP1 that contains threonine 46. The sequence is conserved in human, mouse and rat.
Purification
4E-BP1 antibody was purified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated 4E-BP1. The final product is generated by affinity chromatography using a 4E-BP1-derived peptide that is phosphorylated at threonine 46.
Clonality / Clone
This is a polyclonal antibody.
Host
4E-BP1 [pT46] antibody was raised in rabbit.
Please use anti-rabbit secondary antibodies.
Specificity
Human and mouse 4E-BP1. Rat 4E-BP1 has not been tested.
Application
For Western blotting applications, we recommend using the antibody at a 1:1000 dilution.
Tested Application
WB
Buffer
Rabbit polyclonal immunoglobulin in Dulbecco’s phosphate buffered saline (without Mg2+ and Ca2+), pH 7.3 (+/- 0.1), 50% glycerol with 1.0 mg/mL BSA (IgG, protease free) as a carrier.
Storage
Store at -20˚C. We recommend a brief centrifugation before opening to settle vial contents. Then, apportion into working aliquots and store at -20˚C. For shipment or short-term storage (up to one week), 2-8˚C is sufficient.
Positive Control
- Human embryonic kidney (HEK293) cells stimulated with EGF or 15% serum.
Species Reactivity
H, M
Formulation
Rabbit polyclonal immunoglobulin in Dulbecco’s phosphate buffered saline (without Mg2+ and Ca2+), pH 7.3 (+/- 0.1), 50% glycerol, with 1.0 mg/mL BSA (IgG, protease free) as a carrier.
Short Description
4E-BP1 [pT46]
This product belongs to the following categories:
Related Products
References- Stephens, L., et al. (2005) Phosphoinositide 3-kinases as drug targets in cancer. Curr. Opin. Pharmacol. 5(4):357-365.
- Zhou, L., et al. (2005) 4E-binding protein phosphorylation and eukaryotic initiation factor-4E release are required for airway smooth muscle hypertrophy. Am. J. Respir. Cell Mol. Biol. 33(2):195-202.
- Greenberg, V.L. and S.G. Zimmer (2005) Paclitaxel induces the phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1 through a Cdk1-dependent mechanism. Oncogene 24(30):4851-4860.
- Wang, X., et al. (2005) Distinct signaling events downstream of mTOR cooperate to mediate the effects of amino acids and insulin on initiation factor 4E-binding proteins. Mol. Cell Biol. 25(7):2558-2572.
Datasheet 0801-W
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