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Chuck Reynolds
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What does Resveratrol induce.? P-9, mP-2

Published on 6/23/2019
For additional information  Click Here

Resveratrol induces autophagy by directly inhibiting mTOR through ATP competition P-9


MTT assay

Cells were seeded onto 96-well plates, which were placed in an incubator overnight to allow for attachment and recovery. In brief, cells were treated with 100 μM resveratrol for 48 h and MTT was then dissolved to a concentration of 5 mg/ml in warm assay medium. A total of 20 μl MTT solution was transferred to each well to yield a final volume of 120 μl/well. Plates were incubated for 4 h at 37 °C in 5% CO2. Following incubation, supernatants were removed and 100 μl DMSO was added. Dissolved precipitates were measured with plated reader with wavelength at 540 nm.

In vitro kinase assay and analysis of enzyme kinetics

mTORC1 kinase assay was performed as described previously, with some modifications. Briefly, immunoprecipitated HA-mTOR (kindly provided by Dr. Sabaini, MIT) was washed three times and incubated with kinase assay buffer (20 mM HEPES, 50 mM KCl, 10 mM MgCl2), 200 ng of 4E-BP1 (Stratagene) and the indicated amount of resveratrol for 10 min on ice. The reaction was started with the addition of 50 or 100 μM ATP and was conducted at 30 °C for 10 min. The reaction was stopped by the addition of 5x sample buffer. For a kinase assay using recombinant mTOR, GST-mTOR (Life Technologies) was used and GST-S6K, which is generated by PCR amplification of kinase dead fragment S6K (322 to C-term) and subcloning into pGEX-4T1 vector was purified from E.coli and used as a substrate. An innhibitory curve was drawn using Sigma Plot.

Article Produced By

Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan 689-798, Republic of Korea
Department of Bioscience and Biotechnology, Sejong University, 98 Kunja-Dong, Kwangjin-Ku, Seoul 143-747, Republic of Korea

(All these Articles can be read from this URL: )

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Chuck Reynolds

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