Main description:

Since its discovery, the mammalian target of rapamycin (mTOR) has been shown to regulate many critical molecular processes in eukaryotes such as metabolism, growth, survival, aging, synaptic plasticity, memory, and immunity. In mTOR: Methods and Protocols expert researchers in the field detail many of the methods which are now commonly used to study mTOR. These include methods and techniques used for the study of the mTOR pathway and potential therapeutic applications of mTOR inhibitors such as, immunosuppressive and anticancer agents. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.

Authoritative and practical, mTOR: Methods and Protocols seeks to aid scientists in the further study of this crucially important molecule ranging from the analysis of signal transduction events within a cell to the assessment of complex human diseases such as metabolic disorders or cancer.

Contents:

1. Mammalian Target of Rapamycin: A Signaling Kinase for every Aspect of Cellular Life

Thomas Weichhart

2. Biochemical and Pharmacological Inhibition of mTOR by Rapamycin and an ATP-Competitive mTOR Inhibitor

Ker Yu and Lourdes Toral-Barza

3. Evaluation of the Nutrient-Sensing mTOR Pathway

Sungki Hong, Aristotle M. Mannan and Ken Inoki

4. mTOR Activity Under Hypoxia

Douangsone D. Vadysirisack and Leif W. Ellisen

5. Isolation of the mTOR Complexes by Affinity Purification

Tattym Shaikenov, Olga Bulgakava, Rakhment I. Bersimbaev and Dos D. Sarbassov

6. An in vitro Assay for the Kinase Activity of mTOR Complex 2

Jingxiang Huang

7. Overexpression or Downregulation of mTOR in Mammalian Cells

Mahmoud Khalil and Ivan Gout

8. Dectection of Cytoplasmic and Nuclear Functions of mTOR by Fractionation

Margit Rosner and Markus Hengstschlager

9. Evaluation of Rapamycin-Induced Cell Death

Lorenzo Galluzzi, Eugenia Morselli, Oliver Kepp, Ilio Vitale, Amena Ben Younes, Maria Chiara Maiuri and Guido Kroemer

10. Evaluation of mTOR-Regulated mRNA Translation

Valentina Iadevaia, Xuemin Wang, Zhong Yao, Leonard J. Foster and Christopher G. Proud

11. A Genome-wide RNAi Screen for Polypeptides that Alter rpS6 Phosphorylation

Angela Papageorgiou and Joseph Avruch

12. Immunohistochemical Analysis of mTOR Activity in Tissues

Jinhee Kim, Nancy Otto, Claudio J. Conti, Irma B. Gimenz-Conti and Cheryl L. Walker

13. Assessing Cell Size and Cell Cycle Regulation in Cells with Altered TOR Activity

Megan Cully and Julian Downward

14. Quantitative Visualization of Authophagy Induction by mTOR Inhibitors

Beat Nyfeler, Philip Bergman, Christopher J. Wilson and Leon O. Murphy

15. The in vivo Evaluation of Active-Site TOR Inhibitors (asTORi) in Models of BCR-ABL+Leukemia

Matthew R. Janes and David A. Fruman

16. Inducible raptor and rictor Kncokout Mouse Embryonic Fibroblasts

Nadine Cybulski, Vittoria Zinzalla, and Michael N. Hall

17. Expanding Human T Regulatory Cells with the mTOR- Inbibitor Rapamycin

Manuela Battaglia, Angela Stabilini and Eleonora Tresoldi

18. Rapamycin Induced Enhancement of Vaccine Efficacy in Mice

Chinnaswamy Jagannath and Pearl Bakhru

19. Utilizing a Retroviral RNAi System to Investigate in vivo mTOR Functions in T Cells

Koichi Araki and Bogumila T. Konieczny

20. Exploring Functional in Vivo Consequences of the Selective Genetic Ablation of mTOR Signaling in T Helper Lymphocytes

Greg M. Delgoffe and Jonathan D. Powell

21. Evaluating the Therapeutic Potential of mTOR Inhibitors Using Mouse Genetics

Huawei Li, Jennifer L. Cotton, David A. Guertin

22. Inhibition of PI13K-Akt-mTOR Signaling in Glioblastoma by mTORC1/2 Inhibitors

Qi-Wen Fan and Williams A. Weiss

23. Assessing the Functions of mTOR in Human Embryonic Stem Cells

Jiaxi Zhou, Dong Li, and Fei Wang

24. Video-EEG Monitoring Methods for Characterizing Rodent Models of Tuberous Sclerosis and Epilepsy

Nicholas R. Rensing, Dongjun Guo, and Michael Wong

25. A Genetic Model to Dissect the Role of Tsc-mTORC1 in Neuronal Cultures

Duyu Nie and Mustufa Sahin

26. Tissue-Specific Ablation of Tsc1 in Pancreatic Beta-Cells

Hiroyuki Mori and Kun-Liang Guan

27. A Mouse Model of Diet- Induced Obesity and Insulin REsistance

Chao-Yung Want and James K. Liao

28. Rapamycin as Immunosuppressant in Murine Transplantation Model

Louis-Marie Charbonnier and Alain Le Moine

29. Development of ATP-Competitive mTOR Inhibitors

Qingsong Liu, Seong A. Kang, Carson C. Thoreen, Wooyoung Hur, Jinhua Wang, Jae Won Chang, Andrew Markhard, Jianming Zhang, Taebo Sim, David M. Sabatini and Nathanael S. Gray