46-9718-42
antibody from Invitrogen Antibodies
Targeting: MTOR
FLJ44809, FRAP, FRAP1, FRAP2, RAFT1, RAPT1
Antibody data
- Antibody Data
- Antigen structure
- References [7]
- Comments [0]
- Validations
- Flow cytometry [1]
- Other assay [3]
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Validation data
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- Product number
- 46-9718-42 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-mTOR (Ser2448) Monoclonal Antibody (MRRBY), PerCP-eFluor™ 710, 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 normal human peripheral blood cells. This can be used at 5 µL (0.125 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. PerCP-eFluor® 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome. 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 nm; Emission: 710 nm; Laser: Blue 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 Intestinal microbiota-derived short-chain fatty acids regulation of immune cell IL-22 production and gut immunity.
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.
Requirement for Rictor in homeostasis and function of mature B lymphoid cells.
mTOR signaling in growth control and disease.
Mammalian target of rapamycin protein complex 2 regulates differentiation of Th1 and Th2 cell subsets via distinct signaling pathways.
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
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
Requirement for Rictor in homeostasis and function of mature B lymphoid cells.
Lee K, Heffington L, Jellusova J, Nam KT, Raybuck A, Cho SH, Thomas JW, Rickert RC, Boothby M
Blood 2013 Oct 3;122(14):2369-79
Blood 2013 Oct 3;122(14):2369-79
mTOR signaling in growth control and disease.
Laplante M, Sabatini DM
Cell 2012 Apr 13;149(2):274-93
Cell 2012 Apr 13;149(2):274-93
Mammalian target of rapamycin protein complex 2 regulates differentiation of Th1 and Th2 cell subsets via distinct signaling pathways.
Lee K, Gudapati P, Dragovic S, Spencer C, Joyce S, Killeen N, Magnuson MA, Boothby M
Immunity 2010 Jun 25;32(6):743-53
Immunity 2010 Jun 25;32(6):743-53
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- TOP: Normal human peripheral blood cells were unstimulated (left) or stimulated with Anti-Human CD3 Functional Grade Purified (Product # 16-0037-81) plus Anti-Human CD28 Functional Grade Purified (Product # 16-0289-81) (right) for 48 hours. The cells were then intracellularly stained with Anti-Human CD3 FITC (Product # 11-0036-42) and Anti-Human/Mouse phospho-mTOR (S2448) PerCP-eFluor 710 using the Intracellular Fixation & Permeabilization Buffer Set (Product # 88-8824-00) and protocol. Cells in the lymphocyte gate were used for analysis. BOTTOM: Normal human peripheral blood cells were unstimulated (orange histogram) or were stimulated with Anti-Human CD3 Functional Grade Purified (Product # 16-0037-81) plus Anti-Human CD28 Functional Grade Purified (Product # 16-0289-81) in the presence (green histogram) or absence (purple histogram) of the PI3 kinase inhibitor, LY294002, for 48 hours. The cells were then intracellularly stained with Anti-Human CD3 FITC (Product # 11-0036-42) and Anti-Human/Mouse phospho-mTOR (S2448) PerCP-eFluor 710 using the Intracellular Fixation & Permeabilization Buffer Set (Product # 88-8824-00) and protocol. CD3+ 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.