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Chuck Reynolds
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Chuck Reynolds   My Press Releases

What does Resveratrol induce.? P-9, mP-3

Published on 6/24/2019
For additional information  Click Here

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

Methods-P-3

Immunoprecipitation and Western blotting

HA-mTOR cDNA, HA-Raptor cDNA (kindly provided by Dr. Sabaini, MIT), FLAG-mTOR cDNA (insertion of PCR product from HA-mTOR cDNA into pCMV vector) and FLAG-D2195A mTOR (generated by site directed mutagensis) were transfected with Lipofectamine (Invitrogen) according to the manufacturer’s instructions. Cells were lysed in buffer containing 40 mM HEPES, 120 mM NaCl, 50 mM NaF, 10 mM β-glycerophosphate, 1 mM EDTA, 10 mM sodium pyrophosphate, 1 mM PMSF, 1 μg/μl aprotinin, 2 μg/μl leupeptin and 0.3% CHAPS. Except for the IP samples, 1% TX-100 was used as a detergent. Then 700–1000 μg of protein was incubated with the respective antibody for 4 h followed by incubation with protein A-conjugated agarose beads.

Antibody immobilized beads (Sigma-Aldrich) were used for FLAG-mTOR. Beads were washed three times with the lysis buffer and resuspended with the sample buffer. Prepared samples were subjected to SDS-PAGE (6–16% gradient) and transferred to NC membranes (Amersham Biosciences). The membranes were blocked with TTBS buffer containing 5% skim milk and incubated overnight at 4 °C with antibodies against ULK1 (Santa Cruz Biotechnology), Rictor (Bethyl Laboratories), actin (MP Biomedicals) and other proteins (Cell Signaling). The membranes were next washed with TTBS, incubated with HRP-conjugated secondary antibodies at room temperature for 1 h, and washed again with TTBS. Antibody-bound membranes were developed using ECL (Thermo Fisher).

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

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4763238/

(All these Articles can be read from this URL: 
 
https://markethive.com/group/ama/blog/the-role-of-resveratrol-in-achieving-optimal-health-p5 )

Posted by
Chuck Reynolds

The reasoning for this article is to supply you information about this wonderful product and also for me to find at least two or more people for this simply Amazing Opportunity.. You just need to follow the 4 simple steps..  (Join,Train,sigup 2 or more people and then you duplicate. It's that simple..). This business can make you money in the thousands or just be a customer for a very satisfying experience.

View this video as it helps you to understand a very simple Business Plan. Check it out, then
View these videos (4 of them, very short (total less then 5 min)) that explains how simple
this business works and if so inclined, click on my name above, call me or just sign up
either as a Customer or Distributor.

Thank You
and
Enjoy
Chuck
559-369-7791
chuck@dreams-inn.com
https://chuck212.amazingrape.com/

 

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