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PE Mouse anti-4EBP1 (pT69)
PE Mouse anti-4EBP1 (pT69)

LEFT PANEL: Analysis of 4EBP1 (pT69) in human peripheral blood monocytes. Human peripheral blood mononuclear cells (PBMC) were either treated with 100 μM LY294002 (Sigma, Cat. No. L-9908) for 1 hour at 37ºC (shaded histogram) or untreated (open histogram).  The PBMC were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, and then stained with PE Mouse anti-4EBP1 (pT69).  For data analysis, monocytes were selected by their scatter profile.  The data demonstrates that the level of phosphorylation of 4EBP1 decreases when protein kinase activity is inhibited by the treatment.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

RIGHT PANEL: The specificity of mAb M34-273 was confirmed by western blot analysis (right panel) using unconjugated polyclonal anti-4EBP1 (Cell Signaling Technology, Cat. No. 9542, left blot) and unconjugated monoclonal Mouse anti-4EBP1 (pT69) (right blot) antibodies on lysates from control (lanes 1) and LY294002-treated (lanes 2) PBMC.  4EBP1 is identified as a band of 15-20 kDa in the left blot, regardless of LY294002 treatment.  The right blot demonstrates the reduction of 4EBP1 (pT69) with LY294002 treatment (lane 2).  Purified Mouse anti-Actin monoclonal antibody (Cat. No. 612656 or 612657) was the gel-loading control.

LEFT PANEL: Analysis of 4EBP1 (pT69) in human peripheral blood monocytes. Human peripheral blood mononuclear cells (PBMC) were either treated with 100 μM LY294002 (Sigma, Cat. No. L-9908) for 1 hour at 37ºC (shaded histogram) or untreated (open histogram).  The PBMC were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, and then stained with PE Mouse anti-4EBP1 (pT69).  For data analysis, monocytes were selected by their scatter profile.  The data demonstrates that the level of phosphorylation of 4EBP1 decreases when protein kinase activity is inhibited by the treatment.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

RIGHT PANEL: The specificity of mAb M34-273 was confirmed by western blot analysis (right panel) using unconjugated polyclonal anti-4EBP1 (Cell Signaling Technology, Cat. No. 9542, left blot) and unconjugated monoclonal Mouse anti-4EBP1 (pT69) (right blot) antibodies on lysates from control (lanes 1) and LY294002-treated (lanes 2) PBMC.  4EBP1 is identified as a band of 15-20 kDa in the left blot, regardless of LY294002 treatment.  The right blot demonstrates the reduction of 4EBP1 (pT69) with LY294002 treatment (lane 2).  Purified Mouse anti-Actin monoclonal antibody (Cat. No. 612656 or 612657) was the gel-loading control.

Product Details
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BD Phosflow™
4E-BP1. EIF4EBP1, P/OKCL.6, PHAS-I, PHAS-1
Human (QC Testing)
Mouse BALB/c IgG1, κ
Phosphorylated Human 4EBP1 (pT69) Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645499
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

Either BD Cytofix™ fixation buffer or BD Phosflow™ Fix Buffer I may be used for cell fixation.  Any of the three BD Phosflow™ permeabilization buffers may be used.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
560288 Rev. 2
Antibody Details
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M34-273

The eukaryotic translation initiation factor 4E-Binding Protein 1 (4EBP1) is a phosphorylated heat- and acid-stable protein (PHAS-I or PHAS-1), and it is regulated by insulin. It is a member of the eIF4E-Binding Protein Family, which also includes the proteins 4EBP2 and 4EBP3. 4EBP1 binds with eukaryotic translation Initiation Factor 4E (eIF4E), which prevents its assembly into the eIF4E complex and inhibits cap-dependent translation. When 4EBP1 is phosphorylated, this binding is disrupted, allowing cap-dependent translation to be activated. Phosphorylation of 4EBP1 is required for protein synthesis, and it mediates the regulation of protein translation by stimuli that signal through the phosphoinositide 3 (PI3) kinase pathway. We found that threonine 69 (T69) is phosphorylated in resting human peripheral blood monocytes, but it is almost undetectable in resting lymphocytes. PI3 kinase inhibitors, such as LY294002 down-regulate the phosphorylation level of 4EBP1 (pT69) in monocytes.

The M34-273 monoclonal antibody recognizes the phosphorylated T69 of activated human 4EBP1.  

560288 Rev. 2
Format Details
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PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
560288 Rev.2
Citations & References
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Development References (2)

  1. Gingras AC, Raught B, Gygi SP, et al. Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes Dev. 2001; 15(21):2852-2864. (Biology). View Reference
  2. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev. 2004; 18:1926-1945. (Biology).
560288 Rev. 2

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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.