JAK-STAT Signalling

This Methods in Molecular Biology(TM) volume covers JAK and STAT specific approaches, the negative regulators of the pathway, and production and crystallization of JAK and STAT proteins, and more. Includes protocols, materials lists, tips and troubleshooting advice.

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.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts Includes supplementary material: sn.pub/extras

Contenu

Analysis of Janus Tyrosine Kinase Phosphorylation and Activation.- Co-Immunoprecipitation Protocol to Investigate Cytokine Receptor Associated Proteins e.g. Janus Kinases or Other Associated Signaling Proteins.- In Vitro JAK Kinase Activity and Inhibition Assays.- Quantitative Analysis of JAK Binding using Isothermal Titraion Calorimetry and Surface Plasmon Resonance.- Determination of Protein Turnover Rates in the JAK/STAT Pathway using a Radioactive Pulse-Chase Approach.- Designing RNAi Screens to Identify JAK/STAT Pathway Components.- The Use of JAK-Specific Inhibitors as Chemical Biology Tools.- Methods for Detecting Mutations in the Human JAK2 Gene.- Detection of Activated STAT Proteins.- Detection of Activated STAT Species Using Electrophoretic Mobility Shift Assay (EMSA) and Potential Pitfalls Arising from the Use of Detergents.- Flow Cytometric Analysis of STAT Phosphorylation.- Acetylation of Endogenous STAT Proteins.- Detection and Cellular Localization of Phospho-STAT2 in the Central Nervous System by Immunohistochemical Staining.- Nuclear Trafficking of STAT Proteins Visualized by Live Cell Imaging.- Characterization of STAT Self-Association by Analytical Ultracentrifugation.- Constitutively Active STAT5 Constructs.- Analysis of Suppressor of Cytokine Signalling (SOCS) Gene Expression by Real-Time Quantitative PCR.- Detection of Endogenous SOCS1 and SOCS3 Proteins by Immunoprecipitation and Western Blot Analysis.- In vitro Ubiquitination of Cytokine Signaling Components.- Production and Crystallization of Recombinant JAK Proteins.- Bacterial Expression, Purification and Crystallization of Tyrosine Phosphorylated STAT Proteins.