Cells were lysed by RIPA buffer (100 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, and 1% Triton-X-100) and the cell lysates were harvested by centrifugation at 15,000 rpm for 10 min to remove the debris. The normal brain lysate were purchased from Origene Technologies. Thirty-microgram cell lysates from each group were applied to 10% sodium dodecyl sulfate polyacrylamide gels electrophoresis and proteins were transferred onto polyvinyl difluoride membranes (Millipore, MA, USA). The membrane was blocked with 5% skim milk in TBST for 1 h at room temperature. The antibodies used include anti-TELO2 (Cat. No. ab122722, Abcam, Cambridge, UK), mTOR antibody (cat. No. 2972; Cell Signaling Technology, Beverly, Boston, MA, USA), and β-actin (Santa Cruz Biotechnology, Inc.). Band detection was conducted by enhanced chemi-luminescence and X-ray film (GE Healthcare, Piscataway, NJ, USA).
Mtor antibody
Manufactured by Abcam
Sourced in United Kingdom
The MTOR antibody is a laboratory research tool used to detect and study the mammalian target of rapamycin (mTOR) protein. mTOR is a serine/threonine protein kinase that plays a central role in regulating cell growth, proliferation, and metabolism. This antibody can be used in various applications, such as Western blotting, immunohistochemistry, and immunofluorescence, to investigate the expression and localization of the mTOR protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinyl difluoride membrane, by Merck Group (1 mentions)
Enhanced chemi luminescence and x ray film, by GE Healthcare (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Anti alix, by Abcam (1 mentions)
Zeb1 antibody, by Abcam (1 mentions)
Dnmt3a antibody, by Abcam (1 mentions)
Gapdh antibody, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Roche (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Fusion fx5 spectra, by Vilber (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Pi3k antibody, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibody, by ZSGB-BIO (1 mentions)
Akt antibody, by Cell Signaling Technology (1 mentions)
Avidin biotin complex kit, by Vector Laboratories (1 mentions)
6 protocols using mtor antibody
1
Western Blot Analysis of TELO2, mTOR, and β-Actin
2
Protein Separation and Analysis
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The newly added protease inhibitor and the lysis buffer (SDS) were applied for protein separation. Subsequent to separation through SDS-PAGE, the lysates were transferred onto a polyvinylidene fluoride membrane acquired from Roche. Then the membrane was blocked with 2% BSA, followed by incubation with anti-CD63 and anti-TSG 101 from Santa Cruz, and anti-Alix, ZEB1 antibody, MTOR antibody, DNMT3A antibody and GAPDH antibody from Abcam at 4°C overnight. Then the membrane underwent incubation with the proper secondary antibodies, and the protein expression in cells was measured with GAPDH as an internal reference.
3
Analyzing mTOR Knockout Protein Levels
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Equal amounts of protein extracted from the spleen of tamoxifen induced mTOR–/– mice, mTORflox/floxER-Cre-negative mice, and WT mice were used for electrophoresis and subsequently transferred to polyvinylidene fluoride membrane. The mTOR antibody was diluted at 1:10,000 (Abcam, Cambridge, MA). The normalized β-actin was diluted at 1:1000 (Abcam, Cambridge, MA).
4
Western Blot Analysis of AKT/PI3K/mTOR Pathway
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Whole cell lysates were separated with 10% SDS-PAGE and transferred to nitrocellulose membrane (Millipore, MA). Following being blocked with 5% skimmed milk to block non-specific proteins for 1 h at room temperature, membranes were treated with corresponding primary antibody overnight at 4°C, and then followed by peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies for 1 h at room temperature. Protein bands were visualized using the Tanon High-sig ECL (Tanon Science and Technology Co., Ltd., Shanghai, China) and analyzed with Vilber Fusion Fx5 Spectra (Vilber Lourmat, Marne La Vallée, France). The anti-DEPDC1 antibody was purchased from abcam Inc. (Abcam, Cambridge, MA, USA). GAPDH antibody was obtained from Santa Inc (Dallas, TX, USA). AKT antibody (Cell Signaling Technology, USA), p-AKT (Ser473) (Cell Signaling Technology, USA), PI3K antibody (Santa Cruz, USA), p-PI3K p85 (Tyr458) antibody (Santa Cruz, USA), mTOR antibody (Abcam, USA) and p-mTOR (Ser2448) antibody (Cell Signaling Technology, USA) were used to analyze AKT/PI3K/mTOR signal pathway. HRP-conjugated secondary antibodies were from ZSGB-BIO (Beijing, China).
5
Immunohistochemical Analysis of mTOR Pathway in Retroperitoneal Fibrosis
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Fibrous mass was derived by puncture biopsy or surgery from 16 patients with active RPF. Control samples were the mesenteric root tissue biopsies obtained from four early diagnosed colon cancer patients who were confirmed by pathological examination to have no lymphatic metastasis at the time of biopsy. Tissue samples were fixed and cut into slices of 4 µm thick. After antigen retrieval, non-specific antigen sites were blocked and tissue sections were incubated with mTOR antibodies (Abcam, dilution 1:400), P-AKT (S473) antibodies (Abcam, dilution 1:500) and P-S6K1 (T389+T412) antibodies (Abcam, dilution 1:100). Peroxidase activity was revealed by 3–30-diamino-benzidine-tetrahydrochloride. The fibrotic area was manually outlined, and the software Visiopharm Integrator System was used for quantification.
6
Immunohistochemical Analysis of mTOR in Knee Cartilage
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Knee cartilage from the major load-bearing areas on the MTP and LTP was subjected to immunohistochemical analysis (IHC) using antimammalian target of rapamycin (mTOR) antibodies (Abcam, Cambridge, United Kingdom). Briefly, the cartilage was deparaffinized and rehydrated and then subjected to antigen retrieval by incubating the tissues in sodium citrate buffer (0.01 M, pH 6.0) at 95°C for ten minutes. The tissue sections were exposed to hydrogen peroxide (3% H2O2) for five minutes to quench the endogenous peroxidase and were then blocked in 30% horse serum for 30 minutes. The slides containing the tissue sections were incubated overnight at 4°C with primary mTOR antibodies (1:150 dilution). Nonimmune mouse immunoglobulin G (IgG) was used as a negative control. After the tissues were washed with 1 × tris-buffered saline containing 0.1% Tween-20 (TBST), the slides were then incubated with biotinylated secondary antibodies (antigoat IgG; Santa Cruz Biotechnology, Santa Cruz, California) and detected using an avidin-biotin complex (ABC) kit (Vector Laboratories, Burlingame, California).
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