NF-?B Transcription Factors Methods and Protocols

This detailed book serves as a systematic examination of the analytical methods to study the transcription factor NF-?B in physiology and disease. It provides an up-to-date guidebook to navigate both conventional and highly specialized methods to detect and analyze the different signaling pathways of NF-?B activation and contextualize them within organismal physiology and disease pathogenesis, using genetic and biochemical techniques and some of the most advanced computational and systems biology methods. Additionally, the volume includes several examples of approaches utilized by leading experts in the NF-?B field to analyze and modulate NF-?B signaling in specific physiological and disease contexts, along with some of the most promising approaches to pharmacologically target the NF-?B pathway in human disease. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. 

Part I: General Methods for Analyzing NF-?B Activation

 

1. Measuring NF-kB Phosphorylation and Acetylation

            Nikita Tushar Modi and Lin-Feng Chen

 

2. Biochemical Methods to Analyze the Subcellular Localization of NF- ?B Proteins Using Cell Fractionation

            Davide Vecchiotti, Daniela Verzella, Daria Capece, Mauro Di Vito Nolfi, Barbara Di Francesco, Jessica Cornice, Guido Franzoso, Edoardo Alesse, and Francesca Zazzeroni

 

3. Immunohistochemical Analysis of Expression, Phosphorylation, and Nuclear Translocation of NF-?B Proteins in Human Tissues

            Davide Vecchiotti, Daniela Verzella, Daria Capece, Jessica Cornice, Mauro Di Vito Nolfi, Barbara Di Francesco, Guido Franzoso, Edoardo Alesse, and Francesca Zazzeroni

 

4. High-Throughput Analysis of the Cell- and DNA Site-Specific Binding of Native NF-?B Dimers Using Nuclear Extract Protein-Binding Microarrays (NextPBMs)

            Heather Hook, Rose Zhao, David Bray, Jessica L. Keenan, and Trevor Siggers

 

5. Molecular and Biochemical Approaches to Study the Evolution of NF-?B Signaling in Basal Metazoans

            Pablo J. Aguirre Carrión, Leah M. Williams, and Thomas D. Gilmore

 

Part II: Methods for Studying the Activation of NF-?B Downstream of Distinct Signaling Pathways

 

6. Methods for Modulating the Pathway of NF-kB Using Short Hairpin RNA (ShRNA)

            Marta Moretti, Barbara Di Francesco, Mauro Di Vito Nolfi, Annapaola Angrisani, and Enrico De Smaele

 

7. Immunoblot Analysis of the Regulation of TNF Receptor Family-Induced NF-kB Signaling by c-IAP Proteins

            Eugene Varfolomeev, Tatiana Goncharov, and Domagoj Vucic

 

8. Methods to Study CARD11-BCL10-MALT1 Dependent Canonical NF-kB Activation in Jurkat T-Cells

            Andreas Gewies, Carina Graß, and Daniel Krappmann

 

9. Analysis of Calcium Control of Canonical NF-?B Signaling in B Lymphocytes

            Corbett T. Berry, Michael J. May, and Bruce D. Freedman

 

10. A Kinase Assay for Measuring the Activity of the NIK-IKK1 Complex Induced via         the Noncanonical NF-?B Pathway

            Tapas Mukherjee, Yashika Ratra, Balaji Banoth, Alvina Deka, Smarajit Polley, and Soumen Basak

 

11. Analyze the SUMOylation of IKK?/NEMO During Genotoxic Stress

            Zhao-Hui Wu and Shigeki Miyamoto

 

Part III: Methods for Analyzing NF-?B Activation in Physiology and Disease

 

12. Analysis of the Contribution of NF-kB in the Regulation of Chemotherapy-Induced Cell Senescence by Establishing a Tetracycline-Regulated Cell System

            Francesco Pacifico, Elvira Crescenzi, and Antonio Leonardi

 

13. Methods to Detect NF-?B Activity in Tumor-Associated Macrophage (TAM) Populations

            Fan Sun, Zhaoxia Qu, and Gutian Xiao

 

14. Methods to Study the Effect of IKK Inhibition on TNF-Inducing Apoptosis and Necroptosis in Cultured Cells

            Alessandra Pescatore, Carmela Casale, Francesca Fusco, and Matilde Valeria Ursini

 

15. Use of ChIP-qPCR to Study the Crosstalk between HIF and NF-kB Signaling in Hypoxia and Normoxia

            Dilem Shakir, Michael Batie, and Sonia Rocha

 

16. Methods to Analyze the Roles of TAK1, TRAF6, and NEMO in the Regulation of NF-?B Signaling by RANK Stimulation During Osteoclastogenesis

            Gaurav Swarnkar, Manoj Arra, Suresh Adapala, and Yousef Abu-Amer

 

17. NF-kB Signaling in Ex Vivo Mouse Intestinal Organoids

            Kateryna Shostak, Caroline Wathieu, Sylvia Tielens, and Alain Chariot

 

18. Extracellular Flux Analysis to Investigate the Impact of NF-?B on Mitochondrial Respiration in Colorectal Carcinoma (CRC)

            Daria Capece, Daniela Verzella, Federica Begalli, Jason Bennett, Daniel D’Andrea, Davide Vecchiotti, Francesca Zazzeroni, and Guido Franzoso

 

19. Conditional Knockout Mouse Models to Study the Roles of Individual NF-?B Transcription Factors in Lymphocytes

            Emma J. Adams, Nilushi S. De Silva, and Ulf Klein

 

20. Generation and Surgical Analysis of Genetic Mouse Models to Study NF-?B-Driven Pathogenesis of Diffuse Large B-Cell Lymphoma

            Bernard D. Maybury, Yolanda Saavedra-Torres, Thomas J.A. Snoeks, Jude Fitzgibbon, and Dinis P. Calado

 

21. The Screening of Combinatorial Peptide Libraries for Targeting Key Molecules or Protein-Protein Interactions in the NF-?B Pathway

            Laura Tornatore, Daria Capece, Annamaria Sandomenico, Daniela Verzella, Davide Vecchiotti, Francesca Zazzeroni, Menotti Ruvo, and Guido Franzoso