Technical Data
0004-18J
4E Binding Protein 1, phosphorylated (Thr37/46) (Protein Synthesis Initiation Factor 4E Binding Protein, eIF-4EBP1, PHAS-1, PHAS-I, Eukaryotic Translation Initiation Factor BP)
Description:
When bound to eIF4E, 4E-BP1 (also known as PHAS-1) inhibits cap-dependent translation.
Upon hyperphosphorylation of 4E-BP1 this interaction is disrupted and cap-dependent translation is activated (1). Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase regulate 4E-BP1 activity (2,3). Multiple 4E-BP1 residues are phosphorylated in vivo (4); while phosphorylation by FRAP/mTOR on Thr37 and Thr46 does not prevent the binding of 4E-BP1 to eIF4E, it is thought to prime 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70 (5).

PHAS-I, also known as eIF4E-BP1 and PHAS-II,-III (eIF4E-BP2, 3), are members of a family of proteins that regulate eukaryotic translation initiation which is mediated by the cap structure (m7GpppN, where N=any nucleotide) present at the 5 end of all cellular mRNAs, except organellar (1). The m7 cap is essential for the translation of most mRNA because it directs the translation machinery of the 5 end of the mRNA via its interaction with the cap binding protein, the translation initiation factor 4E(eIF4E) (2). eIF4E plays an principal role in determining global translation rates because its interaction with the cap facilitates the binding of the ribosome to the mRNA. Consistent with this role, eIF4E is required for cell cycle progression, exhibits anti-apoptotic activity and when overexpressed transforms cells (2). Interaction with PHAS proteins prevents incorporation of eIF4E into an active translation initiation complex and inhibits cap-dependent translation. However, this inhibitory effect is alleviated following phosphorylation of the PHAS proteins by a P13K-dependent pathway, involving signaling by the anti-apoptotic kinase Akt/PKB, as well as FRAP/mTOR (2). Rat PHAS-I has 117 amino acids with a apparent molecular weight of 22kD and is 93% identical to eIF-4E-BPI cloned from human placenta (3, 4). PHAS-I and II were found to have overlapping but different patterns of expression in tissues. PHAS-I is expressed in a wide variety of cell types with the highest being in two of the most insulin-responsive tissues, adipocytes and skeletal muscle (3). Both PHAS proteins are phosphorylated in response to insulin or growth factors such as EGF, PDGF and IGF-1. Increasing cAMP in cells promotes dephosphorylation of both PHAS-I and PHAS-II but that regulation of the two protein differ because PHAS-II, unlike PHAS-I is readily phosphorylated by PKA (5). The PHAS-I initiation factor has 28 phosphorylation sites and is multiply phosphorylated by insulin-stimulated protein kinase(s) resulting in 810 phosphorylated isoforms in exponentially growing cells. Changes occur in the expression of these isoforms in response to stresses such as heat shock, and this may contribute to translation repression (6).

Applications:
Suitable for use in Western Blot, Immunohistochemistry, Immunofluorescence, and Flow cytometry. Other applications have not been tested.

Recommended Dilutions:
Western Blot: 1:1000. For Western blots, incubate membrane with diluted antibody in 5% w/v BSA, 1X TBS, 0.1% Tween-20 at 4C with gentle shaking, overnight.
Immunohistochemistry (Paraffin): 1:400 (Citrate/TBST)
Immunofluorescence (IF-IC): 1:50
Flow Cytometry: 1:100
Optimal dilutions to be determined by the researcher.

Storage and Stability:
May be stored at 4C for short-term only. For long-term storage, aliquot and store at -20C. Aliquots are stable for at least 12 months at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
TypeIsotypeCloneGrade
MabIgG9B142Supernatant
SizeStorageShippingSourceHost
100ul-20CBlue IceMouseRabbit
Concentration:
Not determined
Immunogen:
Synthetic phospho-peptide corresponding to residues surrounding Thr37 and Thr46 of mouse 4E-BP1 (KLH-coupled)
Purity:
Supernatant
Form
Supplied as a liquid in 10mM sodium HEPES, pH 7.5, 150mM sodium chloride, 100ug/ml BSA, 50% glycerol, 0.02% sodium azide.
Specificity:
Recognizes endogenous levels of 4E-BP1 only when phosphorylated at Thr37 and/or Thr46. May crossreact with 4E-BP2 and 4E-BP3 when phosphorylated at equivalent sites. Species Crossreativity: human, mouse, rat, monkey, and D. melanogaster.
Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.
(1) Pause, A. et al. (1994) Nature 371, 762767. (2) Brunn, G.J. et al. (1997) Science 277, 99101.
(3) Gingras, A.C. et al. (1998) Genes Dev. 12, 502513. (4) Fadden, P. et al. (1997) J. Biol. Chem. 272, 1024010247. (5) Gingras, A.C. et al. (1999) Genes Dev. 13, 14221437.