The antibody against phosphorylated mTOR (Ser2448) was purchased from Cell Signaling (Davers, MA). The antibody against β-actin was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Protein levels for the phosphorylated mTOR and β-actin in the jejunal mucosa were determined by Western Blot analysis as described previously [22 (link), 23 (link)].
Ser2448
Manufactured by Cell Signaling Technology
Sourced in United States
Ser2448 is a laboratory reagent that can be used to detect the phosphorylation of the serine residue at position 2448 in the mTOR protein. It is a specific antibody that binds to this phosphorylated form of mTOR and can be used in various experimental techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the regulation and activity of the mTOR signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab114071, by Abcam (1 mentions)
Ab91526, by Abcam (1 mentions)
Ab170941, by Abcam (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Ab108349, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Sc 56277, by Santa Cruz Biotechnology (1 mentions)
Sc 8396, by Santa Cruz Biotechnology (1 mentions)
Wblur0500, by GE Healthcare (1 mentions)
Sc 481, by Santa Cruz Biotechnology (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Recombinant human tnf, by R&D Systems (1 mentions)
Luciferase reporter assay system, by Promega (1 mentions)
Antisense and sense oligonucleotides, by Eurofins (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
9 protocols using ser2448
1
Measuring Phosphorylated mTOR in Jejunal Mucosa
2
Comprehensive Western Blot Analysis
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Western blot analysis was performed as previously described.22 (link) Cells were lysed in hot 1× sodium dodecyl sulfate sample buffer, and 30 µg samples were loaded into each lane of 4%–12% polyacrylamide gels. The proteins were separated by electrophoresis, and the proteins in the gels were transferred onto nitrocellulose membranes (Pall; Port Washington, NY, USA). The membrane was blocked with 5% nonfat milk for 1 hour at room temperature and then incubated with antibodies against PR (3176; Cell Signaling Technology, Danvers, MA, USA), pAKT (Ser473; 4058; Cell Signaling Technology), AKT (4685; Cell Signaling Technology), phosphor-mTOR (Ser2448; 5536; Cell Signaling Technology), mTOR (2972; Cell Signaling Technology), LC3B (2775; Cell Signaling Technology), ATG3 (3415; Cell Signaling Technology), ATG5 (8540; Cell Signaling Technology), KRAS (ab137739; Abcam, Cambridge, UK); PTEN (ab170941; Abcam), EIG121 (ab156275; Abcam), P62 (ab91526; Abcam), Beclin-1 (ab114071; Abcam), and β-actin (sc-47778; Santa Cruz Biotechnology Inc., Dallas, TX, USA) for 16 hours at 4°C. Then, the specific horseradish peroxidase-conjugated rabbit anti-mouse or rabbit anti-mouse IgG was added to the membrane and incubated for 1 hour at room temperature. Detection by the chemiluminescence reaction was carried out using the enhanced chemiluminescence (ECL) kit (Pierce; Rockford, IL, USA).
3
Quantifying Tumor mTOR Phosphorylation
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In 6 patients additional mTOR immunohistochemistry was performed as described previously using heat antigen retrieval procedure (citrate buffer) and the phospho-mTOR antibody (Ser-2448, #2976, Cell Signaling Technology, 1:100) according to the manufacturer’s protocol.12 (link) A scoring system was based on the percentage of p-mTOR positive tumor cells and their staining intensity. A score of 180 and above is considered a high mTOR phosphorylation.
4
Protein Expression Analysis Protocol
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Cells were washed with cold PBS and lysed in RIPA buffer (Thermo Fisher Scientific). Protein concentrations were quantified using the BCA protein assay (Thermo Fisher Scientific). Equal amounts of protein were separated on 8‒15% gels using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked for 1 h at room temperature with 5% skim milk diluted in TBS-T. Next, the membrane was probed overnight with primary antibodies against Cyclin E (Santa Cruz #sc-481), CDK2 (Santacruz #sc-748), Cyclin D1 (Santa Cruz #sc-8396), CDK4 (Santa Cruz #sc-56277), GAPDH (Santa Cruz #sc-47724), P16INK4a (Abcam #ab108349), P53 (Abcam #ab32132), P27 (Cell Signaling #2552), mTOR (Cell Signaling #2971), and p-mTOR (Ser2448, Cell Signaling #2532), ATG4A, ATG3) at 4 °C. The membrane was then washed with TBST and incubated with an HRP-conjugated secondary antibody for 1 h at room temperature. The protein bands were visualized using an HRP substrate (Millipore #WBLUR0500) and imaged using an Amersham Imager 600 (GE Healthcare).
5
Signaling Pathways in Inflammatory Response
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Recombinant human TNF was obtained from R&D Systems (Minneapolis, MN). Antibodies were purchased as follows: CXCR2 (E-2, sc-7304) and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA) and IκB, EGFR, Erk1/2, Akt, Raf, MEK, mTOR and their phosphorylated forms such as pIκB (Ser32/36), pEGFR (Tyr1173), pErk1/2 (Thr202/Tyr204), pAkt (Ser473), pB-Raf (Ser445), pc-Raf (Ser338), pMEK (Ser217/221) and pmTOR (Ser2448) were from Cell Signaling Technology (Beverly, MA). Lipofectamine 2000 and all liquid culture media were acquired from Invitrogen (Grand Island, NY). A customized PCR array for the chemokine network, primers for CCL20 and CXCR4, and a SYBR® Green Master Mix came from SABiosciences in Qiagen (Frederick, MD). Chemiluminescent detection kits were from Santa Cruz Biotechnology (Santa Cruz, CA). Antisense and sense oligonucleotides were obtained from Eurofins MWG Operon (Huntsville, AL). Finally, the Luciferase Reporter Assay System was obtained from Promega (Madison, WI).
6
Protein Extraction and Western Blot Analysis
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In brief, proteins were collected using lysis buffer (Cat No. P0013B, Beyotime, Shanghai, China), and the protein concentration was measured using a BCA Protein Assay Kit (PC0020, Solarbio Science & Technology, Beijing, China). Before incubation with the antibody, equal quantities of protein samples were separated by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. A ChemiDoc MP Imaging System was used to image the blots (Bio-Rad, Hercules, CA, USA). p-SAPK/JNK (Thr183/Tyr185; #4685), SAPK/JNK (#9252), p-Src (Ser17; #12432), p-Stat3 (Tyr705; #9145), mTOR (2065AP), p-mTOR (Ser2448; #5536), p-Akt (Ser473; #4060), p-GSK3β (Ser9; #5558), PARP (#9532) and cleaved-PARP (#94885) were provided by Cell Signaling Technology. Src (11097-1-AP), AKT (11097-1-AP), Stat3 (10253-2-AP), ERK1/2 (16443-1-AP), GSK3β (#12456), GAPDH (10494-1-AP), P53 (10442-1-AP), Bcl2 (10068-1-AP), Bax (50599-2-19), IL-6 (21865-1-AP), Nrf2 (16396-1-AP) and HO-1 (10701-1-AP) were purchased from Proteintech. Immobilon® PVDF membranes were provided by Thermo Fisher Scientific (Waltham, MA, USA) and Merck KGaA (Darmstadt, Germany), respectively.
7
Breast Cancer Cell Pathway Analysis
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A total of 20,000 cells/well were grown on 10-mm-diameter coverslips in 24-well plates. After 24 h, the cells were incubated with serum- and steroid-deficient medium and treated with 1 µg/mL tetracycline for 48 h. Then, the cells were treated with solvent (0.1% DMSO (v/v) and 0.1% ethanol (v/v)) as a control and 10 µM Api alone or in combination with 1 nM E2 for 24 h. After treatment, the cells were fixed with 4% formaldehyde solution/neutral buffered saline (Sigma-Aldrich, St Louis, MO, USA) for 15 min and then permeabilized in PBS/3% Triton X-100 for 10 min. Next, the cells were unmasked with 50 mM Tris-HCl pH 9.5 at 80 °C for 30 min. The cells were incubated overnight at 4 °C with primary antibodies against phospho-Bad (Ser112, Cell Signaling Technology, Danvers, MA, USA) (dilution 1:1000), phospho-mTOR (Ser2448, Cell Signaling Technology, Danvers, MA, USA) (dilution 1:1000), FOXO3a (ab124394, Abcam, Cambridge, MA, USA) (dilution 1:1000) or FOXM1 (D12D5, Cell Signaling Technology, Danvers, MA, USA) (dilution 1:1000) and then with a secondary antibody (dilution 1:1000) at room temperature for one hour. The coverslips were mounted with Duolink® in situ mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St Louis, MO, USA) before microscopic analysis.
8
Immunohistochemical Analysis of SSTR-2, GLP-1R, and p-mTOR
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Expression of SSTR-2, GLP-1R, and p-mTOR was evaluated by immunohistochemical staining. Sections (4-mm thick) were cut from paraffin-embedded tissues, deparaffinized in xylene, and rehydrated in graded ethanol, as previously described [21] . Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide in methanol for 30 min. Antigen retrieval was achieved by microwaving the sections in citrate buffer for 20 min. Each section was incubated overnight at 4 C with primary antibodies including rabbit monoclonal antibodies against SSTR-2 (ab134152, dilution 1:100; Abcam, Cambridge, UK) and p-mTOR (Ser2448, #2976, 1:100; Cell Signaling Technology, Danvers, MA, USA) and mouse monoclonal antibodies against GLP-1R (250365, dilution 1:100; ABBIOTEC, San Diego, CA, USA). The sections were sequentially incubated with a biotinylated anti-rabbit or anti-mouse immunoglobulin solution for 20 min. Immunocomplexes were visualized with stable 3, 3 0 -diaminoenzidine tetrahydrochloride (Sigma Aldrich, St Louis, MO, USA) as a chromogen. Sections were then rinsed with distilled water and counterstained with hematoxylin.
9
Western Blot Analysis of mTOR Pathway
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Aliquots of the cells harvested after transfection were mixed with sample buffer, consisting of 0.01% (w/v) bromophenol blue, 2% (w/v) SDS, 5% (w/v) β-mercaptoethanol, 25% (v/v) glycerol and 62.5 mM Tris-HCl (pH 6.8) and boiled for 10 min. Then, 10 µg of protein from the cell samples were loaded onto SDS polyacrylamide gels, electrophoresed and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk for 60 min at room temperature and then incubated overnight at 4°C with the primary antibodies. The primary antibodies were as follows: rabbit anti-Phospho-mTOR (1:1000 dilution; Ser2448, Cell Signaling), rabbit anti-mTOR (1:1000 dilution; Cell Signaling), rabbit anti-Phospho-p70 S6 Kinase (1:1000 dilution; Ser371, Cell Signaling), rabbit anti-Phospho-4E-BP1 (1:1000 dilution; Cell Signaling), rabbit anti-BCL2 (1:2000 dilution; Proteintech) and mouse monoclonal IgG1 antibody specific for β-actin (1:5000 dilution; Santa Cruz Biotechnology). After incubation with the primary antibodies, the membranes were washed four times with Tris-buffered saline containing 0.1% Tween-20 and incubated with the secondary antibodies (1:2000 dilution; Cell Signaling) for 90 min at room temperature. The ECL chemiluminescence kit (Millipore) was used to detect the protein bands.
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