Main description:

Focusing on essential insight into the methods and techniques required to dissect the complex mechanisms of NF- B activation, regulation, and function. NF-kappa B: Methods and Protocols guides readers through standard approaches to detect NF- B pathway activation, detection and analysis of NF- B signaling, and methods to study the control of NF- B signaling. As a volume in the highly successful Methods in Molecular Biology series, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and tips on troubleshooting and avoiding known pitfalls.

Comprehensive and authoritative, NF-Kappa B: Methods and Protocols provides a timely and invaluable resource for researchers seeking to perform experiments aimed at understanding the role of NF- B signaling in health and disease.

Contents:

Part I: Standard Approaches to Detect NF- B Pathway

1. Electrophoretic Mobility Shift Assay Analysis of NF- kB DNA Binding

Sitharam Ramaswami and Matthew S. Hayden M.D

2. Detection of IkB Degradation Dynamics and IkB-a Ubiquitination

Petro Starokadomskyy and Ezra Burstein

3. Measurement of NF- B Transcriptional Activity and Identification of NF- B cis-regulatory Elements using Luciferase Assays

Patricia E. Collins, Christine O'Carroll, and Ruaidhri J. Carmody

Part II: Detection and Analysis of NF- B Signaling

4. Assessing Sites of NF- B DNA Binding using Chromatin Immunoprecipitation

Amy Colleran, Patricia E. Collins, and Ruaidhri J. Carmody

5. IKK Kinase Assay for Assessment of Canonical NF-kB Activation in Neurons

Anca B Mihalas and Mollie K Meffert

6. Analysis of Epidermal Growth Factor-induced NF-kB Signaling

Changying Jiang and Xin Lin

7. Methods to Assess the Activation of the Alternative (non-canonical) NF-kB Pathway by Non-death TNF Receptors

Caroline Remouchamps and Emmanuel Dejardin

8. Systematic Detection of Non-canonical NF- B Activation

Zhaoxia Qu and Gutian Xiao

9. Non-canonical NF- B activation and SDF-1 expression in Human Endothelial Cells

Kelly A. McCorkell and Michael J. May

10. Stable Reconstitution of IKK-deficient Mouse Embryonic Fibroblasts

Carolyn M. Gray and Michael J. May

11. Dissecting NF- B Signaling Induced by Genotoxic Agents Via Genetic Complementation of NEMO-deficient 1.3E2 Cells

Shawn S. Jackson and Shigeki Miyamoto

Part III: Methods to Study the Control of NF- B Signalling

12. Visualizing TCR induced POLKADOTS Formation and NF- B Activation in the D10 T Cell Clone and Mouse Primary Effector T cells

Suman Paul and Brian C Schaefer

13. Detection of Recombinant and Cellular MALT1 Paracaspase Activity

Daniel Nagel and Daniel Krappmann

14. TRAF Protein Function In Non-Canonical NF- B Signaling

Anna D. Reichardt, Jose Pindado Rodriguez, Shivam A. Zaver, Genhong Cheng

15. Roles of c-IAP Proteins in TNF Receptor Family Activation of NF-kB Signaling

Eugene Varfolomeev, Tatiana Goncharov, and Domagoj Vucic

16. Elucidating Dynamic Protein-protein Interactions and Ubiquitination in NF-kB Signaling Pathways

Noula Shembade and Edward W. Harhaj

17. Immunoblot Analysis of Linear Polyubiquitination of NEMO

Yoshiteru Sasaki, Hiroaki Fujita, Misa Nakai, and Kazuhiro Iwai

18. In vitro Detection of NEMO-Ubiquitin Binding using DELFIA and Microscale Thermophoresis Assays

Michelle Vincendeau, Daniel Krappmann, and Kamyar Hadian

19. Use of Fluorescence Spectroscopy For Quantitative Investigations Of Ubiquitin Interactions with the Ubiquitin-Binding Domains of NEMO

Virginie Dubosclard, Elisabeth Fontan, and Fabrice Agou

20. Generation of a Proteolytic Signal: E3/E2-mediated Polyubiquitination of I B

Robert A. Chong, Kenneth Wu, Jordan Kovacev, and Zhen-Qiang Pan

21. Control of NF- B Subunits by Ubiquitination

Patricia E. Collins, Amy Colleran, and Ruaidhri J. Carmody

22. Methodology to Study NF-kB/RelA Ubiquitination in vivo

Haiying Li, Petro Starokadomskyy, and Ezra Burstein

23. Using Sequential Immunoprecipitation and Mass Spectrometry to Identify Methylation of NF-kB

Tao Lu and George R. Stark

24. Methods to Detect NF-kB Acetylation and Methylation

JinJing Chen and Lin-Feng Chen

Part IV : Analyzing and Targeting NF- B Activity in Disease

25. A Method for the Quantitative Analysis of Stimulation-Induced Nuclear Translocation of the p65 subunit of NF-kB from Patient-Derived Dermal Fibroblasts

Alex W. Wessel and Eric P. Hanson

26. Methods for Assessing the In Vitro Transforming Activity of NF- B Transcription Factor c-Rel and Related Proteins

Thomas D. Gilmore and Celine Gelinas

27. Using RNA Interference in Lung Cancer Cells to Target the IKK-NF- B Pathway

Daniela S. Basseres and Albert S. Baldwin

28. Immunohistochemical Analysis of NF-kB in Human Tumor Tissue

Clint T. Allen and Carter Van Waes

29. Assessment of Canonical NF-kB Activity in Canine Diffuse Large B-Cell Lymphoma

Anita Gaurnier-Hausser and Nicola J. Mason

30. NEMO Binding Domain Peptide Inhibition of Inflammatory Signal-Induced NF- B Activation In Vivo

Kelly A. McCorkell and Michael J. May

31. Regulation of NF B Signaling in Osteoclasts and Myeloid Progenitors

Gaurav Swarnkar and Yousef Abu-Amer

32. NF- B Activation with Aging: Characterization and Therapeutic Inhibition

Jing Zhao, Xuesen Li, Sara McGowan, Laura J. Niedernhofer, and Paul D. Robbins

33. The "Sneaking-ligand" Approach: Cell-type Specific Inhibition of the Classical NF-kB Pathway

Bettina Sehnert, Harald Burkhardt, Stefan Dubel, and Reinhard E. Voll

34. Sneaking-ligand Fusion Proteins Attenuate Serum Transfer Arthritis by Endothelium-targeted NF-kB inhibition

Bettina Sehnert, Harald Burkhardt, Michael J. May, Jochen Zwerina, and Reinhard E. Voll

35. Analysis of NF-kB Activation in Mouse Intestinal Epithelial Cells

Helena Shaked, Monica Guma, and Michael Karin

Part V: Bioinformatics and Modeling Approaches to Study NF- B

36. Characterizing the DNA Binding Site Specificity of NF-kB with Protein Binding Microarrays (PBMs)

Trevor Siggers, Thomas D. Gilmore, Brian Barron, and Ashley Penvose

37. Methods for Analyzing the Evolutionary Relationship of NF-kB Proteins Using Free, Web-driven Bioinformatics and Phylogenetic Tools

John Finnerty and Thomas D. Gilmore

38. Studying NFkB Signaling with Mathematical Models

Simon Mitchell, Rachel Tsui, and Alexander Hoffmann