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Western Blotting

Western blot analysis of 4E-BP1 Control Cell Extracts from MCF7 cells, amino acid starved (1 hr), then either untreated (-) or treated with insulin (100 nM, 30 min; +), using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855, Phospho-4E-BP1 (Ser65) (174A9) Rabbit mAb #9456, Phospho-4E-BP1 (Thr70) Antibody #9455, Phospho-4E-BP1 (Ser65) (D9G1Q) Rabbit mAb #13443, and Phospho-4E-BP1 (Thr37/46) Antibody #9459.

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Product Description

As controls, CST recommends using 10 µl of nonphosphorylated and phosphorylated 4E-BP1 cell extracts. Boil for 2 minutes in the original tube, then load 10 μl per mini-gel lane.

Nonphosphorylated 4E-BP1 Control Cell Extracts: Total cell extracts from MCF7 cells, prepared by starving the cells of amino acids for 1 hour, serve as a negative control.

Phosphorylated 4E-BP1 Control Cell Extracts: Total cell extracts from MCF7 cells, prepared by starving the cells of amino acids for 1 hour and then adding back amino acids for 1 hour and treating with insulin for 30 min, serve as a positive control.


Translation repressor protein 4E-BP1 (also known as PHAS-1) inhibits cap-dependent translation by binding to the translation initiation factor eIF4E. Hyperphosphorylation of 4E-BP1 disrupts this interaction and results in activation of cap-dependent translation (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 at 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).


1.  Pause, A. et al. (1994) Nature 371, 762-7.

2.  Brunn, G.J. et al. (1997) Science 277, 99-101.

3.  Gingras, A.C. et al. (1998) Genes Dev 12, 502-13.

4.  Fadden, P. et al. (1997) J Biol Chem 272, 10240-7.

5.  Gingras, A.C. et al. (1999) Genes Dev 13, 1422-37.



For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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4E-BP1 Control Cell Extracts