Phospho mtor ser2481

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-mTOR (Ser2481) is a laboratory reagent used to detect the phosphorylation of the mTOR protein at the Ser2481 site. This antibody can be used in various applications, such as Western blotting, to study the activation and regulation of the mTOR signaling pathway.

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21 protocols using phospho mtor ser2481

Molecular Mechanism Regulation Assay

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N-Hydroxyphthalimide was purchased from Accela ChemBio Co., Ltd. (Shanghai, China). Propidium iodide (PI), RNase A and 4,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich. Antibodies of mTOR, Phospho-mTOR (Ser2448), Phospho-mTOR (Ser2481), S6K1, Phospho-S6K1 (Thr389), Phospho-S6 Ribosomal Protein (Ser235/236), 4E-BP1, Phospho-4E-BP1 (Ser65), Phospho-Akt (Ser473), Phospho-Akt (Thr308), Cleaved PARP, Cleaved Caspase 3, Caspase 9, cyclin B1, cdc2 were obtained from Cell Signaling Technology; antibodies of S6, Akt, P-ERK1/2, β-actin, Caspase 3, Caspase 8, Bcl-xL, survivin were from Santa Cruz; antibody of ERK was from Epitomics; antibody of eIF4E was obtained from BD Biosciences; Alexa Fluor® 647 donkey anti-mouse IgG antibody was purchased from Invitrogen; all the other secondary antibodies were from Sigma-Aldrich.

Wang M., Zhou A., An T., Kong L., Yu C., Liu J., Xia C., Zhou H, & Li Y. (2016). N-Hydroxyphthalimide exhibits antitumor activity by suppressing mTOR signaling pathway in BT-20 and LoVo cells. Journal of Experimental & Clinical Cancer Research : CR, 35, 41.

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Signaling Pathway Profiling in Cancer Cells

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TNF-a was purchased from R&D Systems (catalog #210-TA/CF). ON123300 was obtained from Onconova Therapeutics, Inc. PD0332991, U0126 and QNZ were purchased from Selleckchem. Antibodies directed against PARP (catalog #9542), CASPASES 3 (catalog #9665), 7 (catalog #9492) and 9 (catalog #9502), phospho-Rb^Ser780 (catalog #9307), Rb (catalog #9309), phospho-mTOR^Ser2481 (catalog #2974), mTOR (catalog #4517, phospho-4EBP1 (catalog #9459), 4EBP1 (catalog #9644), phospho-S6RB (catalog #2211), S6RB (catalog #2317), phospho-FOXO1 (catalog #9461), phospho-ERK (catalog #4370), ERK (catalog #9107), phospho-MEK (catalog #9154), MEK (catalog #4694), phospho-NF-κB p65^Ser536 (catalog #3033), and phospho-Rb^Ser807/811 (catalog #9308) were obtained from Cell Signaling Technologies. GAPDH (catalog #SC-47724), AKT (catalog #SC-1618), p130 (catalog #SC-317), CDK4 (catalog #SC-260), CDK2 (catalog #SC-163), CYCLIN D1 (catalog #SC-8396), NF-κB p65 (catalog #sc-8008) and p21-specific (catalog #SC-756) antisera were purchased from Santa Cruz Biotechnology. The phospho-AKTSer473 antibody was purchased from R&D Systems (catalog #AF-887). Phospho-p130 antiserum was obtained from Abcam (catalog #AB68136).

Divakar S.A., Reddy M.V., Cosenza S.C., Baker S.J., Perumal D., Antonelli A.C., Brody J., Akula B., Parekh S, & Reddy E.P. (2015). Dual Inhibition of CDK4/Rb and PI3K/AKT/mTOR Pathways by ON123300 Induces Synthetic Lethality in Mantle Cell Lymphomas. Leukemia, 30(1), 86-93.

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Examining Protein Levels and Cytochrome c Release

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The protein levels in cells were examined using Western blot analysis as described for immunoblotting for LC3. To detect cytochrome c release, the mitochondria-free cytosolic protein fraction was isolated using a mitochondria isolation kit obtained from Thermo Fisher Scientific (Rockford, IL, USA). Antibodies against cytochrome c, mTOR, phospho-mTOR (Ser2481), phospho-mTOR (Ser2448), PARP, Bcl-xL, Bad and Bax were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Bcl-2 and β-actin were obtained from Sigma. The intensity of the immunoreactive bands was determined using GeneTools software (Syngene, Frederick, MD, USA) after scanning the developed films. The results are expressed as the means ± standard deviation (SD) of three independent experiments.

Lin J.F., Tsai T.F., Yang S.C., Lin Y.C., Chen H.E., Chou K.Y, & Hwang T.I. (2017). Benzyl isothiocyanate induces reactive oxygen species-initiated autophagy and apoptosis in human prostate cancer cells. Oncotarget, 8(12), 20220-20234.

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Comprehensive Immunoblotting Analysis of mTOR Pathway

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All the antibodies were from Cell Signaling Technology (Danvers, MA, USA): mTOR (cat #2983), phospho-mTOR (Ser2481, cat #2974), phospho-mTOR (Ser2448, cat #5536), Rictor (cat #9476), phospho-Rictor (Thr1135, cat #3806), Raptor (cat #2280), AKT (cat #4691), phospho-AKT (Ser473, cat #4060), phospho-AKT (Thr308, cat #13038), phospho-AKT (Thr450, cat #9267), GSK-3β (cat #9315), phospho-GSK-3β (Ser9, cat #9336), PTEN (cat #9559), β-actin (cat #4970), 4E-BP1 (cat #9644), phospho-4E-BP1 (Thr37/46, cat #2855), S6K1/p70S6K (cat #9202), phospho-S6K1/phospho-p70S6K (Thr389, cat #9234), Myc-Tag (cat #2276), horseradish peroxidase-linked anti-rabbit IgG (cat #7074), horseradish peroxidase-linked anti-mouse IgG (cat #7076). Lipofectamine LTX, plus reagent, culture medium were from Invitrogen (Carlsbad, CA, USA). Antibiotic–antimycotic, propedium iodide (PI), rapamycin, GSK3β inhibitor (SB212763), PI3K inhibitor (LY294002 and wortmannin) and other chemicals were from Sigma–Aldrich (St Louis, MO, USA). Protease and phosphatase inhibitor cocktails were from Calbiochem (San Diego, CA, USA). Cycle Test Plus kit was from BD Bioscience (East Rutherford, NJ, USA). Super Signal West Pico imaging system was from Thermo Scientific (Rockford, IL, USA).

Bhattacharya K., Maiti S, & Mandal C. (2016). PTEN negatively regulates mTORC2 formation and signaling in grade IV glioma via Rictor hyperphosphorylation at Thr1135 and direct the mode of action of an mTORC1/2 inhibitor. Oncogenesis, 5(5), e227-.

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Detecting mTOR and Its Phosphorylation

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For detection of mTOR, phospho‐mTOR (Ser2448) and phospho‐mTOR (Ser2481; both Cell Signaling Technology), polarized myeloid cells were lysed in radioimmunoprecipitation assay buffer (RIPA buffer, Life Technologies, Carlsbad, CA, USA). Western blot was performed via standard procedure. Densitometric analysis was performed with the Fuji MultiGauge software (Fujifilm).

Wu H., Han Y., Rodriguez Sillke Y., Deng H., Siddiqui S., Treese C., Schmidt F., Friedrich M., Keye J., Wan J., Qin Y., Kühl A.A., Qin Z., Siegmund B, & Glauben R. (2019). Lipid droplet‐dependent fatty acid metabolism controls the immune suppressive phenotype of tumor‐associated macrophages. EMBO Molecular Medicine, 11(11), e10698.

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Western Blot Analysis of Cellular Signaling

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Cells were lysed in a lysis buffer (50 mM Tris pH 7.4, 1% NP40, 150 mM NaCl, 40 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF), and 10 μg/ml of protease inhibitor cocktail (Sigma-Aldrich) for 30 minutes on ice. Protein concentration was determined by the Bradford method (Bio-Rad, Richmond, CA). Total protein content was fractioned by SDS-PAGE and transferred onto nitrocellulose membranes, which were subsequently blocked with 5% nonfat dry milk for 1 hour. Membranes were then incubated overnight with the indicated primary antibodies. Hsp90 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); phospho ERK1/2, ERK1/2 (Thr202/Tyr204), phospho AKT (Ser473), AKT, phospho p70S6K (Thr389), p70S6K, phospho RPS6 (Ser235/Ser236), RPS6, phospho RSK (Ser380), RSK, phospho mTOR (Ser2481), and mTOR antibodies were purchased from Cell Signaling Technologies (Beverly, MA); β-actin was purchased from Sigma-Aldrich. Membranes were incubated with horseradish peroxidase-linked secondary antibodies (GE Healthcare, Buckinghamshire, UK). Protein bands were detected using the ECL system (GE Healthcare). Densitometric analyses were performed using Scion Image software.

Grasso S., Tristante E., Saceda M., Carbonell P., Mayor-López L., Carballo-Santana M., Carrasco-García E., Rocamora-Reverte L., García-Morales P., Carballo F., Ferragut J.A, & Martínez-Lacaci I. (2014). Resistance to Selumetinib (AZD6244) in Colorectal Cancer Cell Lines is Mediated by p70S6K and RPS6 Activation. Neoplasia (New York, N.Y.), 16(10), 845-860.

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Signaling Pathway Antibody Panel

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The following antibodies and reagents were used in this study: phospho-mTOR (Ser2448) (#5536, Cell Signaling), phospho-mTOR(Ser2481) (#2974, Cell Signaling), phospho-AKT (Ser473) (#4058, Cell Signaling), phospho-4E-BP1 (Ser65) (#34443, Cell Signaling), anti-MEK (#9122, Cell Signaling), anti-ERK1/2 (#9102,Cell signaling), phospho-MEK (#9121, Cell Signaling), phospho-ERK1/2 (#9106, Cell Signaling), anti-cleaved PARP (#5625, Cell Signaling), anti-cleaved caspase3 (#9602, Cell Signaling), autophagy sampler kit (#4445, Cell Signaling), and cell cycle regulation kit (#9932T Cell Signaling).

Xu D.Q., Yuan X.J., Toyoda H, & Hirayama M. (2021). Anti-tumor effect of Huaier extract against neuroblastoma cells in vitro. International Journal of Medical Sciences, 18(4), 1015-1023.

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Immunofluorescence Analysis of Phospho-mTOR and TRX

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Immunofluorescence was performed as previously described [47 (link)]. Briefly, cells were grown on glass cover slips for 24 h and treated with or without Andro and NAC as indicated. Cells were then fixed in ice-cold methanol for 5 min, permeabilized with 0.2% Triton X-100 for 10 min, blocked with 6% BSA for 30 min and then incubated with a 1:50 dilution of phospho-mTOR (Ser 2481) (#2974, from Cell Signaling Technology), or TRX (#sc-271281 from Santa Cruz), overnight at 4°C. Cells were then incubated with Alexa Flour 594 labeled goat anti-rabbit IgG (#A11037); or Alexa Flour 488 (#: A-11001) from Thermo Fisher Scientific at 1:200 and DAPI for 1 h [18 (link)]. Sample images were taken at 200 × magnification using an inverted fluorescence microscope (Olympus IX-7, Pennsylvania, USA Fluorescence intensity was quantified using ImageJ software version 1.39 (NIH). RGB composite images were created using Axion Vision rel, 4.6 and analyzed. Cells from five different fields were used for statistical analysis as previously described.

Banerjee A., Banerjee V., Czinn S, & Blanchard T. (2017). Increased reactive oxygen species levels cause ER stress and cytotoxicity in andrographolide treated colon cancer cells. Oncotarget, 8(16), 26142-26153.

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Antibody-Based Protein Analysis Protocol

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Commercially available antibodies to HRP-conjugated p53, cyclin D1, CDK2, TIM23, LC3, p62, phospho-p53 (Ser392 and Ser46), p19-ARF, cMyc, Caspase 8, DRAM1, and LAMP2 were purchased from Santa Cruz Biotechnology, CA. Phospho-MDM2 (Ser166 and Ser186), total MDM2, cleaved PARP, total PARP, pro-caspase 9, Caspase 3, phospho-Drp1 (Ser616), Beclin1, phospho-Ulk1 (Ser757), phospho-AMPKα (Thr172), phospho-Beclin1 (Ser15), and phospho-mTOR (Ser2481) were procured from Cell Signaling Technology, MA. HRP-conjugated antibody to actin, Mdivi1, Z-VAD-fmk, chloroquine, and FH535 were purchased from Sigma-Aldrich, MO. LAPTM4B was purchased from Novus Biologicals, CO. Commercially available AZD5363, rapamycin, and mTOR inhibitor (AZD8055) were procured from Cayman Chemicals, MI.

Patra T., Meyer K., Ray R.B, & Ray R. (2020). A combination of AZD5363 and FH5363 induces lethal autophagy in transformed hepatocytes. Cell Death & Disease, 11(7), 540.

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Signaling Pathway Profiling in Cancer Cells

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