Cell Signaling Technology

Product Pathways - Translational Control

4E-BP Antibody Sampler Kit #9955

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855 40 µl W IHC-P IF-IC F H M R Mk Dm 15 to 20 Rabbit IgG
Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb #4923 40 µl W F H M R Mk 15-20 Rabbit IgG
Phospho-4E-BP1 (Ser65) Antibody #9451 40 µl W IP H M R Mk 15 to 20 Rabbit
4E-BP1 (53H11) Rabbit mAb #9644 40 µl W IP IHC-P IF-IC F H M R Mk 15-20 Rabbit IgG
4E-BP2 Antibody #2845 40 µl W IP IHC-P F H M R Mk B 15 to 20 Rabbit
Phospho-4E-BP1 (Thr70) Antibody #9455 40 µl W IP H M R Mk 15 to 20 Rabbit
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Dm=D. melanogaster  B=Bovine
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Phospho-4E-BP1 (Thr37/46) Rabbit mAb detects endogenous levels of 4E-BP1 only when phosphorylated at Thr37 and/or Thr46, and may cross-react with 4E-BP2 and 4E-BP3 when phosphorylated at equivalent sites. Nonphospho-4E-BP1 (Thr46) (87D12) Rabbit mAb 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 4E-BP1 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.

Western Blotting

Western Blotting

Western blot analysis of bacterially expressed GST-4E-BP1 and of extracts from NIH/3T3 cells using 4E-BP2 Antibody #2845 and 4E-BP1 Antibody #9452.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293 cells using 4E-BP1 Antibody #9452 (upper) and Phospho-4E-BP1 (Thr37/46) Antibody #2855 (lower). The cells were starved for 24 hours in serum-free medium and underwent a 1 hour amino acid depravation. Amino acids were then added back for 1 hour, and cells were either untreated (-) or treated with 100 nM insulin (+) for 30 minutes.

Western Blotting

Western Blotting

Western blot analysis of extracts from COS cells, λ phosphatase-treated or 20% FBS-treated, using Nonphospho-4E-BP1 (Thr46) (87D12) Rabbit mAb #4923 (upper), Phospho-4E-BP1 (Thr37/46) Antibody #9459 (middle), and 4E-BP1 Antibody #9452 (lower).


Western Blotting

Western Blotting

Western blot analysis of extracts from C6 and MEF cells using Phospho-4E-BP1 (Ser65) Antibody #9451 (upper) or 4E-BP1 (53H11) Rabbit mAb #9644 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell types using 4E-BP1 (53H11) Rabbit mAb #9644.

Description

The 4E-BP Antibody Sampler Kit provides an economical means to investigate regulation of cap-dependent translation within the cell. The kit contains primary and secondary antibodies to perform four Western blots with each antibody.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr37 and Thr46 of mouse 4E-BP1, residues surrounding Thr46 of human 4E-BP1, or Ser112 of human 4E-BP1. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the residues at the carboxy-terminus of human 4E-BP2 (#2845), or phosphopeptides surrounding mouse Ser65 (#9451) and human Thr70 (#5078) 4E-BP1. Polyclonal antibodies were purified by protein A and peptide affinity chromatography.

Background

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).

4E-BP2 and 4E-BP3 share high sequence homology with 4E-BP1, including conservation of the major FRAP/mTOR-dependent phosphorylation sites. Preliminary data suggests that phosphorylation of 4E-BP2 is regulated in a similar manner to that of 4E-BP1, although phosphorylation of this protein has not been as extensively studied (6).

  1. Pause, A. et al. (1994) Nature 371, 762-767.
  2. Brunn, G.J. et al. (1997) Science 277, 99-101.
  3. Gingras, A.C. et al. (1998) Genes Dev. 12, 502-513.
  4. Fadden, P. et al. (1997) J. Biol. Chem. 272, 10240-10247.
  5. Gingras, A.C. et al. (1999) Genes Dev. 13, 1422-1437.
  6. Lin, T.A. and Lawrence, J.C. (1996) J. Biol. Chem. 271, 30199-30204.

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