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- BD OMICS-Guard Sample Preservation Buffer
- BD Rhapsody™ ATAC-Seq Assays
- BD Rhapsody™ TCR/BCR Next Multiomic Assays
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LEFT PANEL: Analysis of 4EBP1 (pT36/pT45) 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 (pT36/pT45). 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 M31-16 was confirmed by western blot analysis using unconjugated polyclonal anti-4EBP1 (Cell Signaling Technology, Cat. No. 9542, left blot) and unconjugated monoclonal Mouse anti-4EBP1 (pT36/pT45) (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 (pT36/pT45) with LY294002 treatment (lane 2). Purified Mouse anti-Actin monoclonal antibody (Cat. No. 612656 or 612657) was the gel-loading control.
BD™ Phosflow PE Mouse anti-4EBP1 (pT36/pT45)
BD™ Phosflow PE Mouse anti-4EBP1 (pT36/pT45)
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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.
제품 고시
- 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).
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
관련 제품
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 threonines 36 and 45 (T36/T45) are phosphorylated in resting human peripheral blood mononuclear cells. PI3 kinase inhibitors, such as LY294002 down-regulate the phosphorylation level of 4EBP1 (pT36/pT45).
The M31-16 monoclonal antibody recognizes the phosphorylated T36 and T45 of activated human 4EBP1. The orthologous phosphorylation sites in mouse and rat 4EBP1 are T35 and T44.
개발 참고 자료 (2)
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Gingras AC, Raught B, Gygi SP, et al. Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes Dev. 2001; 15(21):2852-2864. (Biology). 참조 보기
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Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev. 2004; 18:1926-1945. (Biology).
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