JAK-STAT Signalling Methods and Protocols

About this book

The receptor-associated JAK protein kinases and their substrates, the STAT transcriptional activators, transmit signals following cytokine and growth factor binding to receptors expressed on the cell surface, to result in specific transcriptional and cellular responses. Over the last two decades, the field has progressed from identification of the individual components through to an understanding of the activation and deactivation mechanisms, and the complex structural detail of the proteins involved. We now know that these pathways are important in many biological processes, including growth and development, hematopoiesis, and the innate and adaptive immune response. JAK-STAT Signalling: Methods and Protocols provides detailed methodologies for examining many aspects of the pathway. Divided into four sections, topics include JAK and STAT specific approaches, the negative regulators of the pathway (SOCS proteins), and the production and crystallization of JAK and STAT proteins, among others.

Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, JAK-STAT Signalling: Methods and Protocols will be of use not only to those working in the area but also to new investigators who are led to delve into the complexities of JAK and STAT responses.

Contents

Part I. JAK Protein Tyrosine Kinases
1. Analysis of Janus Tyrosine Kinase Phosphorylation and Activation Jeremy A. Ross, Rodriguez Georgialina, and Robert A. Kirken
2. Co-Immunoprecipitation Protocol to Investigate Cytokine Receptor Associated Proteins e.g. Janus Kinases or Other Associated Signaling Proteins Claude Haan and Serge Haan
3. In Vitro JAK Kinase Activity and Inhibition Assays Jeffrey J. Babon and James M. Murphy
4. Quantitative Analysis of JAK Binding using Isothermal Titraion Calorimetry and Surface Plasmon Resonance Jeffrey J. Babon
5. Determination of Protein Turnover Rates in the JAK/STAT Pathway using a Radioactive Pulse-Chase Approach Anna Dittrich, Elmar Siewert, and Fred Schaper
6. Designing RNAi Screens to Identify JAK/STAT Pathway Components Katherine H. Fisher, Stephen Brown, and Martin P. Zeidler
7. The Use of JAK-Specific Inhibitors as Chemical Biology Tools Christopher J. Burns, David Segal, and Andrew F. Wilks
8. Methods for Detecting Mutations in the Human JAK2 Gene Anthony J. Bench, E. Joanna Baxter, and Anthony R. Green

Part II. Signal Transducers and Activators of Transcription (STAT)s
9. Detection of Activated STAT Proteins Jean-Patrick Parisien and Curt M. Horvath
10. Detection of Activated STAT Species Using Electrophoretic Mobility Shift Assay (EMSA) and Potential Pitfalls Arising from the Use of Detergents Serge Jaan and Claude Haan
11. Flow Cytometric Analysis of STAT Phosphorylation Jane Murphy and Gabrielle L. Goldberg
12. Acetylation of Endogenous STAT Proteins Torsten Ginter, Thorsten Heinzel, and Oliver H. Kramer
13. Detection and Cellular Localization of Phospho-STAT2 in the Central Nervous System by Immunohistochemical Staining Reza Krotooshi and Trevor Owens
14. Nuclear Trafficking of STAT Proteins Visualized by Live Cell Imaging Velasco Cimica and Nancy C. Reich
15. Characterization of STAT Self-Association by Analytical Ultracentrifugation Nikola Wenta and Uwe Vinkemeier
16. Constitutively Active STAT5 Constructs Lynn M. Heltemes-Harris and Michael A. Farrar

Part III. Suppressor of Cytokine Signalling (SOCS) Proteins
17. Analysis of Suppressor of Cytokine Signalling (SOCS) Gene Expression by Real-Time Quantitative PCR Tatiana B. Kolesnik and Sandra E. Nicholson
18. Detection of Endogenous SOCS1 and SOCS3 Proteins by Immunoprecipitation and Western Blot Analysis Jian-Guo Zhang and Sandra E. Nicholson
19. In vitro Ubiquitination of Cytokine Signaling Components Jeffrey J. Babon, Artem Laktyushin, and Nadia J. Kershaw

Part IV. Structural Analysis of the JAK/STAT Pathway
20. Production and Crystallization of Recombinant JAK Proteins Isabelle S. Lucet and Rebecca Bamert
21. Bacterial Expression, Purification and Crystallization of Tyrosine Phosphorylated STAT Proteins Florence Baudin and Christoph W. Muller

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