Lung tissue from each mouse was sampled three times and prepared for western blot. The lung tissues were lysed in protein lysis buffer, and proteins were extracted and quantified by Coomassie blue staining. Proteins were detected by western blot using antibodies against p-PI3K (Cell Signaling Technology), p-Akt (Cell Signaling Technology), p-mTOR (Santa Cruz Biotechnology), and p-p70S6k (Epitomics). Proteins were quantified using Odyssey software 3.0 and normalized against β-actin as an internal control.
P mtor
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany, Japan
P-mTOR is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed to measure the phosphorylation state of the mammalian target of rapamycin (mTOR) protein, a key regulator of cell growth, proliferation, and metabolism. The core function of P-mTOR is to provide researchers with a reliable and accurate method to assess the activation status of the mTOR signaling pathway in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (18 mentions)
P akt, by Santa Cruz Biotechnology (16 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (10 mentions)
Bcl 2, by Santa Cruz Biotechnology (10 mentions)
Gapdh, by Santa Cruz Biotechnology (9 mentions)
P p70s6k, by Santa Cruz Biotechnology (5 mentions)
Caspase 3, by Santa Cruz Biotechnology (5 mentions)
P pi3k, by Santa Cruz Biotechnology (5 mentions)
Mmp 2, by Santa Cruz Biotechnology (4 mentions)
P erk, by Santa Cruz Biotechnology (4 mentions)
Murf1, by Santa Cruz Biotechnology (4 mentions)
P erk, by Cell Signaling Technology (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
P akt, by Cell Signaling Technology (3 mentions)
P pi3k, by Cell Signaling Technology (3 mentions)
P nf κb, by Santa Cruz Biotechnology (3 mentions)
Cyclin d1, by Santa Cruz Biotechnology (3 mentions)
Ki 67, by Nichirei Biosciences (3 mentions)
P 4e bp1, by Santa Cruz Biotechnology (3 mentions)
Beclin 1, by Santa Cruz Biotechnology (3 mentions)
53 protocols using p mtor
1
Lung Protein Signaling Pathway Analysis
2
Western Blot Analysis of Vascular Proteins
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Total protein from VSMCs and rat carotid arteries tissues was isolated using RIPA buffer with protease inhibitor Cocktail (Pierce, Rockford, IL, USA). Protein content was measured with a BCA Protein Assay Kit (Cwbiotech, Beijing, China). The equal amounts of proteins were resolved by 10% SDS-PAGE (Sigma Aldrich, St. Louis, MO) and then transferred onto polyvinylidene difluoride (PVDF) membranes (BD Pharmingen, San Diego, CA). After blocking with 5% nonfat milk at room temperature for 1 h, the PVDF membranes were incubated primary antibodies against PCNA (Proteintech Group, Wuhan, China), PTEN, p-AKT, AKT, p-mTOR, mTOR and β-actin (Santa Cruz, CA, USA) at 4°C overnight. All primary antibodies diluted 1:1000. The goat anti-mouse IgG horseradish peroxidase secondary antibody (diluted 1:5000) was purchased from Santa Cruz Biotechnology, Inc. Subsequently, the protein bands were scanned on the X-ray film using the enhanced chemiluminescence detection system (PerkinElmer Life and Analytical Sciences, Boston, MA). The alpha Imager software (Alpha Innotech Corporation, San Leandro, CA) was used to measure relative intensity of each band on western blots. The measurements were conducted independently for at least three times with similar results.
3
Protein Expression Analysis via Western Blot
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Total proteins were extracted using 100 μl lysis buffer form cells. After measurement of the quality and concentrations of the proteins, 25 μg samples were loaded and separated in 10% sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes through electroblotting. Then membranes were blocked with 5% skim milk for 1 h at room temperature, followed by incubation with P-gp (Cat. No. MA5-13854, Invitrogen, Carlsbad, CA, USA), MRP1 (SC-18835), Survivin (sc-17779), MMP2 (sc-13594), pSer473-Akt (sc-293125), Akt1 (sc-5298), p-ERK (sc-7383), ERK (sc-514302), pSer33/37-β-catenin (sc-57535), β-catenin (sc-7963), p-NFκB (sc-271908), NFκB (sc-8414), p-mTOR (sc-293089), mTOR (sc-517464), and GAPDH (SC-47724) antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4 °C in a dilution of 1:1000. Membranes were washed three times with phosphate-buffered saline Tween-20 and incubated with HRP-conjugated secondary antibody (Merck & Co., Inc., Kenilworth, NJ, USA) for another 1 h in a dilution of 1:2500. Immunoreactivity was detected using the Western Lighting Ultra (Thermo Fisher Scientific, Waltham, MA, USA).
4
Extraction and Characterization of Cedrus atlantica
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On a small-scale, extract of Cedrus atlantica (CAt) from Morocco was prepared in our laboratory. The bark of CAt (600g) was extracted by steam-distillation with a flow rate of approximately 7.2 ml/min at 100~105℃ for 90 mins. On a large-scale, preparation of CAt extract was commissioned to Phoenix (New Jersey, USA) following the above conditions. CAt extract and temozolomide (purity ≥ 99%, International Laboratory USA, CA, USA) were dissolved in dimethyl sulfoxide (DMSO) before each experiment, and a final concentration of DMSO was < 0.5% in experiments of cells treatment.
An antibody against p-H2A.X was purchased from Cell Signaling Technology (Beverly, MA, USA), p-ATR, p-Chk2 were purchased from Biorbyt Ltd. (Cambridge, UK), and β-actin were purchased from iReal Biotechnology (Hsinchu, Taiwan). Antibodies used to detect p-ATM, p-Chk1, p53, p-p53, p21, p-RB CDK4, cyclin D1, CDK2, cyclin A, cyclin B1, PCNA, Fas, Fas-L, Bax, Bcl-2, AIF, caspase-8, caspase-9, caspase-3, AKT, p-AKT, mTOR, p-mTOR, P70S6K and p-P70S6K were purchased from Santa Cruz Biotechnology (CA, USA). The detail information of antibodies were descripted in Table1 .
An antibody against p-H2A.X was purchased from Cell Signaling Technology (Beverly, MA, USA), p-ATR, p-Chk2 were purchased from Biorbyt Ltd. (Cambridge, UK), and β-actin were purchased from iReal Biotechnology (Hsinchu, Taiwan). Antibodies used to detect p-ATM, p-Chk1, p53, p-p53, p21, p-RB CDK4, cyclin D1, CDK2, cyclin A, cyclin B1, PCNA, Fas, Fas-L, Bax, Bcl-2, AIF, caspase-8, caspase-9, caspase-3, AKT, p-AKT, mTOR, p-mTOR, P70S6K and p-P70S6K were purchased from Santa Cruz Biotechnology (CA, USA). The detail information of antibodies were descripted in Table
5
Investigating Breast Cancer Cell Signaling
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MCF-7 and MDA-MB-231 cells were obtained from ATCC (Manassas, VA). DMEM/F12, fetal bovine serum (FBS), Lipofectamine TM Reagent was purchased from Invitrogen (Paisley, UK). The anti pAkt, Akt, pmTOR, mTOR, GAPDH and horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). The anti Beclin 1, P62, ILC3, Caspase 3, Bcl2 and Bax were purchased from Proteintech (Wuhan, Hubei, China). Enhanced chemiluminescence (ECL) assay kit was purchased from Amersham (Louisville, Co).
6
Investigating Akt/mTOR Signaling Pathway
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7
Nobiletin Isolation and Characterization
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Nobiletin was prepared from a polymethoxyflavonoid mixture, which was provided by Zhejiang Quzhou Tiansheng Plant Extraction Co. Ltd. in China, containing ~60% nobiletin and tangeretin. The polymethoxyflavonoid mixture was dissolved in methanol-dimethyl sulfoxide (1:1), its concentration was 50 mg/ml, then chromatographed with high-performance liquid chromatography (HPLC) (Waters) eluted with methanol-H2O (70:30) in 8 ml/min at room temperature, separated into two fractions, collected individually, evaporated, obtained fraction I and fraction II. Fraction I was identified as nobiletin (Fig. 1A ) by HPLC-MS, UV-vis chromatography and comparing peak time with that of nobiletin sample from Sigma and previous reports (data not shown). Its purity was >98%. Monoclonal antibodies against HIF-1α, NF-κB (p50), PTEN, c-Myc, GAPDH, p-AKT, total AKT, p-mTOR and total mTOR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The secondary antibodies of anti-rabbit and anti-mouse were purchased from Thermo Scientific (Pierce, Rockford, IL, USA). The Hif -1α and mAkt plasmid constructs were obtained from Addgene (www.addgene.org ) (28 (link)).
8
Protein Expression Analysis by Western Blot
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Cells were seeded in a 6-well plate at a density of 1×105 cells/well for 24 h. To isolate total protein, 250 µl RIPA buffer (Biosdec, Wuhan, China) was added to each well for 30 min. Expression of associated proteins was analyzed using a western blot kit (Biosdec, Wuhan, China). Protein samples (50 µg) were separated by SDS-PAGE gel electrophoresis and transferred to a 0.45 µm polyvinylidene fluoride membrane followed by incubation overnight at 4°C with rat anti-human antibodies [light chain 3B (LC3B), phosphatidylinositol 3-kinase (PI3K), Akt, phosphorylated (p)-Akt, mammalian target of rapamycin (mTOR), p-mTOR, and β-actin] (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:1,000. After decoloration, the samples were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Biosdec, Wuhan, China) at a 1:5,000 dilution for 1 h at room temperature and unbound antibody was washed away. Proteins were visualized following treatment with enhanced chemiluminescence reagent (Biosdec, Wuhan, China) for 1–2 min and exposure to X-ray film. Developed films were processed using BandScan software (Glyko Inc., Novato, CA, USA) to determine optical densities.
9
Western Blot Analysis of Protein Signaling
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Total cellular proteins were extracted with radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology, Jiangsu, China). The protein concentration was measured using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). Briefly, equivalent quantities of protein sample (40 µg/lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently electrotransferred onto polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Subsequently, the membrane was blocked at 4°C for 1 h with TBS containing 0.05% Tween-20 (TBST) buffer with 5% non-fat milk and then incubated with the following primary antibodies against HMGA2 (1:1,000; cat. no. ab97276; Abcam), AKT (1:1,000; cat. no. sc-8312; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), phosphorylated (p)-AKT (1:1,000; cat. no. sc-33437; Santa Cruz Biotechnology, Inc.), mammalian target of rapamycin (1:500, mTOR; cat. no. sc-8319; Santa Cruz Biotechnology, Inc.) and p-mTOR (1:500, cat. no. sc-101738; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Following incubation with secondary antibodies conjugated with horseradish peroxidase (LK2001 and LK2003; 1:100; Sungene Biotech, Co., Ltd., Tianjin, China) for 1 h at room temperature, the bands were examined using an enhanced chemiluminescence system (MultiSciences Biotech Co., Ltd., Hangzhou, China).
10
Western Blot Analysis of Autophagy Proteins
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The cells were lysed by a protein extraction kit (KeyGEN, Nanjing, China). The concentrations of protein were measured by BCA kit. The proteins were separated by SDS gel electrophoresis and transferred to a polyvinylidene difuoride membrane for 2.5 hours. The membrane was blocked with 5% skim milk for 1 hour at room temperature. The membrane was incubated at 4°C overnight with rabbit monoclonal anti-human LC3 (Santa Cruz Biotechnology, Dallas, TX, United States of America), P62 (Cell Signaling Technology, Beverly, MA, USA), Beclin1 (Proteintech, Manchester, United Kingdom), AMP-activated protein kinase (AMPK) (Cell Signaling Technology, Beverly, MA, United States of America), p-AMPK (Cell Signaling Technology, Beverly, MA, USA), mammalian target of rapamycin (mTOR) (Santa Cruz Biotechnology, Dallas, TX, United States of America), p-mTOR (Santa Cruz Biotechnology, Dallas, TX, United States of America), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Dallas, TX, United States of America) primary antibodies, and incubated with a secondary antibody at room temperature for 1 hour.
An enhanced chemiluminescence kit (Bio-Rad, Hercules, CA, United States of America) was used to visually detect proteins.
An enhanced chemiluminescence kit (Bio-Rad, Hercules, CA, United States of America) was used to visually detect proteins.
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