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 ñII 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).

PHAS-I initiation factor has 2–8 phosphorylation sites and is multiply phosphorylated by insulin-stimulated protein kinase(s) resulting in 8-10 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).

Suitable for use in Immunofluorescence, Flow Cytometry, ELISA, Western Blot, Immunoprecipitation, Immunohistochemistry, Immunocytochemistry. Other applications not tested.

Western Blot: 1:1000 Optimal dilutions to be determined by the researcher.

May be stored at 4°C for short-term only. For long-term storage, aliquot and store at -20°C. Aliquots are stable for at least 12 months at -20°C. 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.

Purified by Protein A and peptide affinity chromatography.

Not determined

Supplied as a liquid in 10mM sodium HEPES, pH 7.5, 150mM sodium chloride, 0.1mg/ml BSA, 50% glycerol.

Detects endogenous levels of 4E-BP1 only when dephosphorylated at Thr46. This antibody cross-reacts with 4E-BP2 and 4E-BP3 dephosphorylated at equivalent sites. Phospho- 4E-BP1 (Ser65) antibody detects endogenous levels of 4EBP1 when phosphorylated at Ser65, and may also recognize 4E-BP1 when phosphorylated at Ser101. Phospho-4E-BP1 (Ser65) (174A9) rabbit Mab detects endogenous levels of 4E-BP1 when phosphorylated at Ser65. Phospho-4E-BP1 (Thr70) antibody detects endogenous levels of 4E-BP1 only when phosphorylated at Thr70. 4E-BP1 (53H11) rabbit Mab detects endogenous levels of total 4E-BP1 protein. 4E-BP2 antibody detects endogenous levels of total 4E-BP2 independent of phosphorylation and does not cross-react significantly with 4E-BP1.

This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.