Revision 1
Cell Signaling Technology

Orders: 877-616-CELL (2355) orders@cellsignal.com

Support: 877-678-TECH (8324)

Web: info@cellsignal.com cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IF-IC, FC-FP

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

15-20

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q13542, #Q13541, #O60516

Entrez-Gene Id:

1979, 1978, 8637

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunofluorescence (Immunocytochemistry) 1:200
Flow Cytometry (Fixed/Permeabilized) 1:200

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb detects endogenous levels of 4E-BP1 only when dephosphorylated at Thr46. The antibody cross-reacts with 4E-BP2 and 4E-BP3 dephosphorylated at equivalent sites.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr46 of human 4E-BP1.

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).

  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.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 1
#4923

Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb

Western Blotting Image 1: Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb Expand Image
Western blot analysis of extracts from COS cells, treated with λ phosphatase or 20% FBS as indicated, using Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb (upper), Phospho-4E-BP1 (Thr37/46) Antibody #9459 (middle), and 4E-BP1 Antibody #9452 (lower).
Immunofluorescence Image 1: Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of 293 shRNA Scramble cells either serum starved (far left); serum starved and treated with U0126 #9903 (10 μM, 2 hr), LY294002 #9901 (50 μM, 2 hr), and Rapamycin #9904 (10 nM, 2 hr) (center, left); serum starved and treated with l phosphatase (10,000 U/mL, 2 hr) ) (center, right); and 293 shRNA 4E-BP1/2 KO treated with U0126 #9903 (10 μM, 2 hr), LY294002 #9901 (50 μM, 2 hr), and Rapamycin #9904 (10 nM, 2 hr) (far right), using Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb (green). Red = Propidium Iodide (PI)/RNase Staining Solution.
Flow Cytometry Image 1: Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb Expand Image
Flow cytometric analysis of COS cells, untreated (blue) or λ phosphatase-treated (green), using Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb compared to a nonspecific negative control antibody (red).