AMPK Methods and Protocols

Contents

Preface. .................................................................... v
Contributors................................................................. xi

1 Production and Crystallization of Full-Length Human AMP-Activated

Protein Kinase (α 1 β 1 γ1)................................................. 1

Julia A. Hubbard, Bing Xiao, and Jon R. Wilson

2 Visualizing AMPK Drug Binding Sites Through Crystallization

of Full-Length Phosphorylatedα 2 β 1 γ1 Heterotrimer....................... 15

Christopher G. Langendorf, Jonathan S. Oakhill, and Bruce E. Kemp

3 Biophysical Interactions of Direct AMPK Activators. ........................ 29

Ravi G. Kurumbail, Graham M. West, Venkatasubramanian

Dharmarajan, Kris A. Borzilleri, Jane M. Withka, Jessica Ward,

Allan R. Reyes, Francis Rajamohan, Patrick R. Griffin,

and Matthew F. Calabrese

4 Biochemical Measurement of Glycogen: Method to Investigate

the AMPK-Glycogen Relationship.. ...................................... 57

Elite Possik and Arnim Pause

5 Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK ......... 69

Fiona A. Fyffe, Simon A. Hawley, Alexander Gray,

and D. Grahame Hardie

6 Applications of NMR and ITC for the Study of the Kinetics

of Carbohydrate Binding by AMPKβ-Subunit Carbohydrate-Binding

Modules.. ............................................................. 87

Paul R. Gooley, Ann Koay, and Jesse I. Mobbs

7 Bioinformatics Approach to Identify Novel AMPK Targets................... 99

Brendan Gongol, Traci Marin, David A. Johnson,

and John Y. -J. Shyy

8 Studying AMPK in an Evolutionary Context ............................... 111

Arpit Jain, Valentin Roustan, Wolfram Weckwerth,

and Ingo Ebersberger

9 AMPK Protein Interaction Analyses by Yeast Two-Hybrid................... 143

Pascual Sanz, Rosa Viana, and Maria Adelaida Garcia-Gimeno

10 Transient Expression of AMPK Heterotrimer Complexes
in Mammalian Cells ..................................................... 159
Jonathan S. Oakhill, John W. Scott, and Toby A. Dite

11 Knockdown of Human AMPK Using the CRISPR/Cas
Genome-Editing System................................................. 171
Adrien Grenier, Pierre Sujobert, Se ́verine Olivier, He ́le`ne Guermouche,
Johanna Monde ́sir, Olivier Kosmider, Benoit Viollet,
and Je ́roˆme Tamburini

12 Compound C/Dorsomorphin: Its Use and Misuse as an AMPK Inhibitor..... 195
Biplab Dasgupta and William Seibel

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