Jak-Stat Signalling: Methods and Protocols

ISBN-13: 9781627032414 / Angielski / Twarda / 2013 / 320 str.

Sandra E. Nicholson; Nicos Anthony Nicola

Opis

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(TM) 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.

Spis treści

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. Krämer

 

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. Müller

Blurb

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.