12-9107-42

antibody from Invitrogen Antibodies

Targeting: EIF4EBP1 4E-BP1, PHAS-I

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

12-9107-42

Provider

Invitrogen Antibodies

Product name

Phospho-4EBP1 (Thr36, Thr45) Monoclonal Antibody (V3NTY24), PE, eBioscience™

Antibody type

Monoclonal

Antigen

Other

Description

Description: The V3NTY24 monoclonal antibody recognizes human and mouse eukaryotic translation initiation factor eIF4E-binding protein 1 (4E-BP1) when phosphorylated at threonine 37 and/or threonine 46. 4E-BP1 is a member of a family of translation repressor proteins that include 4E-BP2 and 4E-BP3. In its non-phosphorylated form, 4E-BP1 binds to the eIF4E translation initiation factor and represses cap-dependent translation. Phosphorylation of 4E-BP1 at multiple sites is necessary to disrupt this interaction and de-repress cap-dependent translation. Studies have identified several kinases that phosphorylate 4E-BP1. For instance, FRAP/mTOR phosphorylates Thr37 and Thr46, while ATM phosphorylates Ser111. Phosphorylation of 4E-BP1 at Thr37 and Thr46 is inhibited by the PI3 kinase inhibitors LY294002 and wortmannin. Applications Reported: This V3NTY24 antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This V3NTY24 antibody has been pre-titrated and tested by intracellular staining and flow cytometric analysis of stimulated normal human peripheral blood cells. This can be used at 5 µL (0.06 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. Protocols: Use of Protocol A: Two-step protocol for intracellular (cytoplasmic) proteins is recommended and allows for the greatest flexibility for detection of surface and intracellular (cytoplasmic) proteins. Protocol C: Two-step protocol: Fixation/Methanol may be used and allows for the greatest discrimination of phospho-specific signaling between unstimulated and stimulated samples, but with limitations on the ability to stain specific surface proteins (refer to "Clone Performance Following Fixation/Permeabilization" located in the Best Protocols Section under the Resources tab online). Use of Protocol B: One-step protocol for intracellular (nuclear) proteins is not recommended. All Protocols can be found in the "Staining Intracellular Antigens for Flow Cytometry Protocol" located in the Best Protocols Section under the Resources tab online.. Excitation: 488-561 nm; Emission: 578 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser. Filtration: 0.2 µm post-manufacturing filtered.

Reactivity

Human, Mouse

Host

Mouse

Conjugate

Yellow dye

Isotype

IgG

Antibody clone number

V3NTY24

Vial size

100 Tests

Concentration

5 µL/Test

Storage

4° C, store in dark, DO NOT FREEZE!

References

Submitted references

Autophagy-dependent glutaminolysis drives superior IL21 production in HIV-1-specific CD4 T cells.

Loucif H, Dagenais-Lussier X, Avizonis D, Choinière L, Beji C, Cassin L, Routy JP, Fritz JH, Olagnier D, van Grevenynghe J
Autophagy 2022 Jun;18(6):1256-1273

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IFNα Impairs Autophagic Degradation of mtDNA Promoting Autoreactivity of SLE Monocytes in a STING-Dependent Fashion.

Gkirtzimanaki K, Kabrani E, Nikoleri D, Polyzos A, Blanas A, Sidiropoulos P, Makrigiannakis A, Bertsias G, Boumpas DT, Verginis P
Cell reports 2018 Oct 23;25(4):921-933.e5

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Sestrin2 Suppresses Classically Activated Macrophages-Mediated Inflammatory Response in Myocardial Infarction through Inhibition of mTORC1 Signaling.

Yang K, Xu C, Zhang Y, He S, Li D
Frontiers in immunology 2017;8:728

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Intracellular staining of 30-minute LY294002-treated (left), or 30-minute LPS-stimulated (right) normal human peripheral blood cells with Anti-Human CD14 FITC (Product # 11-0149-42) and 0.06 µg of Anti-Human/Mouse phospho-4E-BP1 (T36/T45) PE (right) using the Intracellular Fixation and Permeabilization Buffer Set (Product # 88-8824-00) and protocol. Total viable cells were used for analysis.

Conjugate

Yellow dye

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 3 Impaired Autolysosomal Degradation Determines the Enhanced Immunogenic Potential of IFNalpha-Shaped and SLE Monocytes CD14 + monocytes from healthy donors were cultured for 18 hr with IFNalpha (400 ng/mL) +/- rapam. (1 muMu) as depicted. (A) Levels of HLA-DR and CD86 membrane expression were measured by flow cytometry. A representative result is depicted. Geometric mean fluorescence intensity (GeoMFI) averages are plotted (n = 6). (B) Concentrations of secreted IL6 and TNFalpha measured by ELISA in culture supernatants (n = 3). (C) CFSE-labeled cord blood-naive CD4 + T cells were cultured for 6 days with allogeneic monocytes, previously treated for 18 hr with IFNalpha, +/- rapam., and analyzed for their proliferation. Proliferation index averages of 4 experiments are graphed. Right: histograms of CFSE dilution and size (FS) of CD4 + gated cells. GeoMFI of un-proliferated cells and FS counting are listed on right side of histograms. One representative result is depicted. (D) p-4EBP1 and p-P70S6K expression in freshly isolated monocytes from healthy (n = 5) and SLE (n = 5) donors were analyzed by flow cytometry, and averages of their GeoMFIs are graphed. (E) MLR between SLE monocytes +- rapam. (1 muM) for 1 hr, cocultured with allogeneic CFSE-labeled cord blood-naive CD4 + T cells for 6 days (n = 3). Averages of proliferation indexes as in (C) and averages of % HLADR hi CD86 hi population after cocultures are graphed. (F) Representative histogram of Lysosensor Gre

Conjugate

Yellow dye