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.
Boil for 3 minutes prior to use. Load 10 μl of phosphorylated and nonphosphorylated Smad2/3 Control Cell Extracts per lane.
Store at -20°C. Supplied in SDS Sample Buffer: 62.5 mM Tris- HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red.
Nonphosphorylated 4E-BP1 Control Cell Extracts: Total cell extracts from MCF7 cells, amino acids starved for 1 hour to serve as a negative control. Supplied in SDS Sample Buffer.
Phosphorylated 4E-BP1 Control Cell Extracts: Total cell extracts from MCF7 cells, amino acids starved for 1 hour followed by adding back amino acids for 1 hour and treating with 100 nM insulin for 30 min to serve as a positive control. Supplied in SDS Sample Buffer.
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).
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