25-9718-42
antibody from Invitrogen Antibodies
Targeting: MTOR
FLJ44809, FRAP, FRAP1, FRAP2, RAFT1, RAPT1
Antibody data
- Antibody Data
- Antigen structure
- References [6]
- Comments [0]
- Validations
- Flow cytometry [1]
- Other assay [3]
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Validation data
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- Product number
- 25-9718-42 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-mTOR (Ser2448) Monoclonal Antibody (MRRBY), PE-Cyanine7, eBioscience™
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- Description: This MRRBY monoclonal antibody recognizes human and mouse mammalian target of rapamycin (also known as mTOR, FRAP, RAFT) when phosphorylated on S2448. mTOR is a serine/threonine protein kinase that functions as an ATP and amino acid sensor as well as to balance nutrient availability with cell growth, proliferation, motility, survival, protein synthesis, and transcription. Activated mTOR increases production of enzymes necessary for glycolysis and controls the uptake of glucose and other nutrients. Increased glucose uptake and metabolism helps fulfill the energy needs for mTOR-driven cell growth and proliferation. When sufficient nutrients are available, mTOR transmits a positive signal to p70 S6 kinase and participates in the inactivation of the eIF4E inhibitor, 4E-BP1. mTOR is phosphorylated at S2448 via the PI3 kinase/Akt signaling pathway and is autophosphorylated at Ser2481. Due to its critical role in regulation of cell growth, survival, and metabolism, and becauseit is often abnormally regulated in tumors, mTOR is under investigation as a potential target for anti-cancer therapy. Applications Reported: This MRRBY antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This MRRBY antibody has been pre-titrated and tested by intracellular staining followed by 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. Staining Protocol: All protocols work well for this monoclonal antibody. Use of Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins allows for the greatest flexibility for detection of surface and intracellular (cytoplasmic) proteins. Use of Protocol B: One-step protocol: intracellular (nuclear) proteins is recommended for staining of transcription factors in conjunction with surface and phosphorylated intracellular (cytoplasmic) proteins. Protocol C: Two-step protocol: Fixation/Methanol 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). All Protocols can be found in the Flow Cytometry Protocols: "Staining Intracellular Antigens for Flow Cytometry Protocol" located in the Best Protocols Section under the Resources tab online. Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light. Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. Excitation: 488-561 nm; Emission: 775 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser. Filtration: 0.2 µm post-manufacturing filtered.
- Reactivity
- Human, Mouse
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- MRRBY
- Vial size
- 100 Tests
- Concentration
- 5 µL/Test
- Storage
- 4° C, store in dark, DO NOT FREEZE!
Submitted references Lung tumor MHCII immunity depends on in situ antigen presentation by fibroblasts.
Intestinal microbiota-derived short-chain fatty acids regulation of immune cell IL-22 production and gut immunity.
The Bone Marrow Protects and Optimizes Immunological Memory during Dietary Restriction.
mTOR-Dependent Oxidative Stress Regulates oxLDL-Induced Trained Innate Immunity in Human Monocytes.
Store-Operated Ca(2+) Entry Controls Clonal Expansion of T Cells through Metabolic Reprogramming.
mTORC1-dependent metabolic reprogramming is a prerequisite for NK cell effector function.
Kerdidani D, Aerakis E, Verrou KM, Angelidis I, Douka K, Maniou MA, Stamoulis P, Goudevenou K, Prados A, Tzaferis C, Ntafis V, Vamvakaris I, Kaniaris E, Vachlas K, Sepsas E, Koutsopoulos A, Potaris K, Tsoumakidou M
The Journal of experimental medicine 2022 Feb 7;219(2)
The Journal of experimental medicine 2022 Feb 7;219(2)
Intestinal microbiota-derived short-chain fatty acids regulation of immune cell IL-22 production and gut immunity.
Yang W, Yu T, Huang X, Bilotta AJ, Xu L, Lu Y, Sun J, Pan F, Zhou J, Zhang W, Yao S, Maynard CL, Singh N, Dann SM, Liu Z, Cong Y
Nature communications 2020 Sep 8;11(1):4457
Nature communications 2020 Sep 8;11(1):4457
The Bone Marrow Protects and Optimizes Immunological Memory during Dietary Restriction.
Collins N, Han SJ, Enamorado M, Link VM, Huang B, Moseman EA, Kishton RJ, Shannon JP, Dixit D, Schwab SR, Hickman HD, Restifo NP, McGavern DB, Schwartzberg PL, Belkaid Y
Cell 2019 Aug 22;178(5):1088-1101.e15
Cell 2019 Aug 22;178(5):1088-1101.e15
mTOR-Dependent Oxidative Stress Regulates oxLDL-Induced Trained Innate Immunity in Human Monocytes.
Sohrabi Y, Lagache SMM, Schnack L, Godfrey R, Kahles F, Bruemmer D, Waltenberger J, Findeisen HM
Frontiers in immunology 2018;9:3155
Frontiers in immunology 2018;9:3155
Store-Operated Ca(2+) Entry Controls Clonal Expansion of T Cells through Metabolic Reprogramming.
Vaeth M, Maus M, Klein-Hessling S, Freinkman E, Yang J, Eckstein M, Cameron S, Turvey SE, Serfling E, Berberich-Siebelt F, Possemato R, Feske S
Immunity 2017 Oct 17;47(4):664-679.e6
Immunity 2017 Oct 17;47(4):664-679.e6
mTORC1-dependent metabolic reprogramming is a prerequisite for NK cell effector function.
Donnelly RP, Loftus RM, Keating SE, Liou KT, Biron CA, Gardiner CM, Finlay DK
Journal of immunology (Baltimore, Md. : 1950) 2014 Nov 1;193(9):4477-84
Journal of immunology (Baltimore, Md. : 1950) 2014 Nov 1;193(9):4477-84
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Normal human peripheral blood cells were unstimulated (left) or stimulated with Anti-Human CD3 and CD28 Functional Grade Purifieds (Product # 16-0037-81 and Product # 16-0289-81) for 48 hours (right). The cells were then intracellularly stained with Anti-Human CD3 APC (Product # 17-0036-42) and Anti-Human/Mouse phospho-mTOR (S2448) PE-Cyanine7 using the Intracellular Fixation & Permeabilization Buffer Set (Product # 88-8824-00) and protocol. Cells in the lymphocyte gate were used for analysis.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- NULL
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Fig. 5 Stat3 and mTOR regulate IL-22 production by CD4 + T cells. a-d WT CD4 + T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions with or without butyrate (0.5 mM) ( n = 3/group). Phosphorylated Stat3 (6 h) ( a , b ) and phosphorylated mTOR (24 h) ( c , d ) were assessed by western blot and flow cytometry. Phosphorylated S6K was analyzed by flow cytometry ( e ). f - i CBir1 Tg CD4 + T cells were activated with APCs and CBir1 peptide under Th1 conditions with butyrate (0.5 mM) +- rapamycin (1 uM) or HJC0152 (1 uM). IL-22 mRNA ( f ) and protein ( g ) were assessed by qRT-PCR and ELISA at 60 h ( n = 3/group). Expression of Hif1a ( h ) and Ahr ( i ) was analyzed at 48 h by qRT-PCR. j WT and Stat3 -/- CD4 + T cells were treated with or without butyrate (0.5 mM) for 5 days ( n = 3/group). IL-22 production was measured by flow cytometry. One representative of three independent experiments was shown. Data were expressed as mean +- SD. Statistical significance was tested by two-tailed unpaired Student t -test ( a - e , j ) or two-tailed one-way ANOVA ( f - i ). a * p = 0.0134; b *** p = 0.0002; c ** p = 0.0059; d *** p = 0.0002; e ** p = 0.0010; f , **** p < 0.0001; g ** p = 0.0019 (butyrate vs control) and 0.0069 (butyrate + rapamycin vs butyrate), * p = 0.0141; h *** p = 0.0004, ** p = 0.0012; i , *** p = 0.0004 (butyrate vs control) and 0.009 (butyrate + HJC0152 vs butyrate), ** p = 0.0030; j ** p = 0.0017, * p = 0.0338.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 6 Antioxidants inhibit activation of the mTOR-HIF1alpha-axis. Monocytes were pre-incubated for 1 h with 0.5 muM Diphenyleneiodonium (DPI), 25 muM VAS2870 or 40 muM Mito-TEMPO or vehicle and treated with oxLDL or vehicle for 24 h. Phosphorylation of mTOR and HIF1alpha accumulation was assessed by staining with PE-Cyanine7 anti-human p-mTOR and PE anti-human HIF1alpha Antibody and analyzed by FACS on day 1 (A,B) or day 6 (C,D) . The MFI (mean fluorescence intensity) was compared between experimental groups. (E) Cells were pre-incubated for 1 h with 40 muM Mito-TEMPO or vehicle and treated with oxLDL or vehicle for 24 h. Lactate concentration was measured on day 6 cells using a colorimetric assay kit. Graphs represent mean values +- SD of at least 6 individuals in at least 3 different experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001.