NF-κB Transcription Factors: Methods and Protocols

Dedication
Preface
Contents
Contributors
Part I: General Methods for Analyzing NF-κB Activation
Chapter 1: Measuring NF-κB Phosphorylation and Acetylation
1 Introduction
2 Materials
2.1 Cell Lines, Primary Cells and Tissues
2.2 Antibodies
2.3 Recombinant Proteins
2.4 Radioisotope
2.5 Buffers and Reagents
3 Methods
3.1 Phosphorylation of RelA
3.1.1 In Vitro Phosphorylation Assay
3.1.2 In Vivo Phosphorylation of RelA S536
Detection of Phosphorylated Overexpressed RelA (Co-transfection with IKK2)
Detection of Phosphorylation of Endogenous RelA in BMDMs
Detection of Chromatin-Associated Phosphorylated RelA by Chromatin Immunoprecipitation (ChIP)
3.2 Acetylation of RelA
3.2.1 In Vitro Acetylation Assay
3.2.2 In Vivo Acetylation Assay
Detection of Acetylation of Overexpressed RelA by Immunoblotting
Detection of Acetylation of Endogenous RelA in BMDMs
Detection of Acetylated Endogenous RelA by Immunohistochemistry (IHC)
Detection of Acetylated RelA by Immunocytochemsitry (ICC)
4 Notes
References
Chapter 2: Biochemical Methods to Analyze the Subcellular Localization of NF-κB Proteins Using Cell Fractionation
1 Introduction
2 Materials
2.1 Lysis Buffer for Nuclear and Cytoplasmatic Extracts
3 Methods
3.1 NF-κB Nuclear and Cytoplasmic Extracts from Adherent and Suspension Cells
3.1.1 Adherent Cell Culture Preparation and Cytoplasmic Proteins Extraction
3.1.2 Suspension Cell Culture Preparation and Cytoplasmic Proteins Extraction
3.1.3 Nuclear Protein Extraction from Adherent and Suspension Cells
4 Notes
References
Chapter 3: Immunohistochemical Analysis of Expression, Phosphorylation, and Nuclear Translocation of NF-κB Proteins in Human T...
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 NF-κB IHC on FFPE Tissue Section
3.1.1 Tissue Preparation
3.1.2 Deparaffinization and Rehydration
3.1.3 Antigen Retrieval: HIER
3.1.4 Inactivation of Endogenous Peroxidases
3.1.5 Blocking Non-specific Antibody Binding Sites
3.1.6 Antibodies Incubation
3.1.7 Detection and Nucleus Counterstain
3.1.8 Dehydration and Mounting
3.1.9 Visualization
3.2 NF-κB IHC on Frozen-OCT Embedded Tissue Section
3.2.1 Tissue Preparation
3.2.2 Inactivation of Endogenous Peroxidases
3.2.3 Blocking Nonspecific Antibody Binding Sites
3.2.4 Antibodies Incubation
3.2.5 Detection and Nucleus Counterstain (See Note 22)
3.2.6 Dehydration and Mounting
3.2.7 Visualization
4 Notes
References
Chapter 4: High-Throughput Analysis of the Cell and DNA Site-Specific Binding of Native NF-κB Dimers Using Nuclear Extract Pro...
1 Introduction
2 Materials
2.1 Preparation of Cell Nuclear Extracts
2.2 Double Stranding Microarray Probes
2.3 Nuclear Extract Protein-Binding Microarray (nextPBM) Experiment
3 Methods
3.1 Preparation of Cell Nuclear Extracts
3.1.1 Cell Harvesting for Adherent Cells
3.1.2 Cell Harvesting for Suspension Cells
3.1.3 Nuclear Extraction
3.2 Double Stranding Microarray Probes
3.3 Nuclear Extract Protein-Binding Microarray (nextPBM) Experiment
3.4 Data Analysis
4 Notes
References
Chapter 5: Molecular and Biochemical Approaches to Study the Evolution of NF-κB Signaling in Basal Metazoans
1 Introduction
2 Materials
2.1 Codon Optimization of DNA Sequences for Expression in Tissue or Bacterial Cells
2.2 Creation of Tissue Lysates
2.3 Western Blotting
2.4 Electrophoretic Mobility Shift Assay
2.5 Immunohistochemistry of Whole Mount and Sectioned Nematostella vectensis Tissue
2.6 Cnidocyte Staining
2.7 Immunohistochemistry of Sectioned Sponge Tissue
3 Methods
3.1 Codon Optimization of cDNA Sequences for Expression in Tissue Culture or Bacterial Cells
3.2 Creation of Tissue Lysates for Western Blotting and EMSAs
3.2.1 Sponge Lysates
3.2.2 N. vectensis Lysates
3.2.3 Aiptasia Lysates
3.2.4 Coral Lysates
3.3 Western Blotting of Protein From Animal Lysates (See Note 8)
3.3.1 Preparing an SDS-Polyacrylamide Gel
3.3.2 Preparing and Running Samples for Western Blotting
3.3.3 Western Blotting
3.4 EMSA Using Animal Lysates
3.4.1 Radiolabeling κB Site Probe
3.4.2 DNA-Binding Reaction and Running EMSA Gel
3.5 Immunohisto chemistry of Whole Mount N. vectensis (Nv) Tissue
3.5.1 Fixation of Anemones
3.5.2 Indirect Immunofluorescence
3.6 Immunohistochemistry of Sectioned N. vectensis Tissue
3.7 Cnidocyte Staining in N. vectensis (See Note 1)
3.8 Immunohistochemical Staining of Sectioned Sponge Tissue
4 Notes
References
Part II: Methods for Studying the Activation of NF-κB Downstream of Distinct Signaling Pathways
Chapter 6: Methods for Modulating the Pathway of NF-κB Using Short Hairpin RNA (ShRNA)
1 Introduction
2 Materials
2.1 Generation of the Lentiviral-ShRNA Vector
2.2 Calcium-Phosphate Transfection and Lentivirus Production
2.3 Lentiviral Transduction
3 Methods
3.1 Generation of ShRelA-Carrying Lentivirus
3.1.1 Design a Stem-Loop Sequence for pLenti Lox 3.7-GFP
3.1.2 Cloning pLenti Lox3.7-GFP Vectors for shRelA
3.2 Lentivirus Production
3.3 Virus Titration
3.4 Lentiviral Transduction of Mammalian Cells
3.5 Selection of Stably Infected Clones
3.6 Identification of the Cells Which Present Efficient Gene Silencing
4 Notes
References
Chapter 7: Immunoblot Analysis of the Regulation of TNF Receptor Family-Induced NF-κB Signaling by c-IAP Proteins
1 Introduction
1.1 Regulation of NF-κB Signaling by c-IAP Proteins
2 Materials
2.1 Equipment
2.2 Reagents
2.2.1 Materials for Western Blotting
2.2.2 Antibodies
3 Methods
3.1 Western Blot-Based Assessment of Activation of MAPKs and NF-κB Signaling Pathways by TNF Ligands
3.1.1 siRNA Transfection
3.1.2 Cell Growth and BV6 Treatment
3.1.3 Treatment of Cells with TNF Ligands or Agonistic Anti-TNF Receptor Antibodies
3.1.4 Cell Lysis and Protein Sample Preparation
3.1.5 SDS-PAGE and Membrane Transfer
3.1.6 Immunoblotting
3.2 Subcellular Fractionation of Proteins
3.3 Immunoprecipitation of Endogenous Receptor-Associated Protein Signaling Complexes
3.3.1 Secondary Immunoprecipitation of Ubiquitinated Proteins in Receptor Signaling Complexes
4 Notes
References
Chapter 8: Methods to Study CARD11-BCL10-MALT1 Dependent Canonical NF-κB Activation in Jurkat T Cells
1 Introduction
2 Materials
2.1 CRISPR/Cas9 Genomic Editing
2.2 Lentiviral Transduction
2.3 Cell Stimulation and NF-κB-EGFP Reporter
3 Methods
3.1 Generation of CARD11 KO Jurkat T Cells by CRISPR/Cas9
3.2 Lentiviral Transduction of Jurkat T Cells
3.2.1 Transduction of the NF-κB-EGFP Reporter Construct into Jurkat T Cells
3.2.2 Lentiviral Reconstitution of CARD11 KO;NF-κB-EGFP Jurkat T Cells Clones
3.3 Investigating NF-κB Activity in Knockout and Reconstituted Jurkat T Cell Clones
4 Notes
References
Chapter 9: Analysis of Calcium Control of Canonical NF-κB Signaling in B Lymphocytes
1 Introduction
2 Materials
2.1 B Lymphocyte Isolation and Stimulation
2.2 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.3 Sample Preparation and Blotting for IκBα Phosphorylation and Degradation
2.4 Confocal and Immunocytochemical Analysis of p65 and c-Rel Localization
2.5 Quantitative RT-PCR Analysis of NF-κB Target Gene Expression
3 Methods
3.1 B Lymphocyte Isolation and Stimulation (See Note 2 About T Cells)
3.2 SDS-PAGE
3.3 Analysis of Phospho-IκBα and IκB (Fig. 1b, See Note 13)
3.4 Blotting and Immunostaining
3.5 Immuno-Fluorescence Analysis of p65 and c-Rel Localization (Fig. 1d)
3.6 Measurement of NF-κB Driven Gene Expression (Fig. 1e)
4 Notes
References
Chapter 10: A Kinase Assay for Measuring the Activity of the NIK-IKK1 Complex Induced via the Noncanonical NF-κB Pathway
1 Introduction
2 Materials
2.1 Cell Culture
2.1.1 Primary Cells and Cell Lines
2.1.2 Cell Culture Media and Supplements
2.2 Reagents and Standard Biochemical Assays
2.2.1 Ligands and Kinase Substrates
2.2.2 Chemicals and Buffers
2.2.3 Antibodies
2.2.4 Biochemical Assays Buffers
2.2.5 Equipments
3 Methods
3.1 Cell Culture
3.2 Treatment with Ligands
3.3 Cell Lysate Preparation
3.3.1 For Adherent Cells (MEFs, BLS4, BLS12, and BMDMs)
3.3.2 For Suspension Culture (B Cells or HMCLs)
3.4 Preparing Cell Lysate for Immunoprecipitation
3.5 Immunoprecipitation of Cellular NIK
3.6 In Vitro Kinase Assay
3.7 SDS-PAGE and Autoradiography
3.8 Immunoblotting
3.9 Summary
4 Notes
References
Chapter 11: Analyze the SUMOylation of IKKγ/NEMO During Genotoxic Stress
1 Introduction
2 Materials
3 Methods
4 Notes
References
Part III: Methods for Analyzing NF-κB Activation in Physiology and Disease
Chapter 12: Analysis of the Contribution of NF-κB in the Regulation of Chemotherapy-Induced Cell Senescence by Establishing a ...
1 Introduction
2 Materials
2.1 Establishment of a Tetracycline-Regulated Cell System
2.2 Induction of Premature Senescence
2.3 Senescence-Associated β-Galactosidase (SA-β-gal) Staining
2.4 BrdU Staining of Cells for Flow Cytometric Analysis
2.5 Detection of DNA Damage Foci by Immunofluorescence Microscopy
2.6 Conditioned Media Preparation
3 Methods
3.1 Establishment of a Tetracycline-Regulated Cell System
3.2 Induction of Premature Senescence
3.3 Senescence-Associated β-Galactosidase (SA-β-gal) Staining
3.4 BrdU Staining of Cells for Flow Cytometric Analysis
3.5 Detection of DNA Damage Foci by Immunofluorescence Microscopy
3.6 Conditioned Media Preparation
4 Notes
References
Chapter 13: Methods to Detect NF-κB Activity in Tumor-Associated Macrophage (TAM) Populations
1 Introduction
2 Materials
2.1 Immunohistochemistry (IHC)
2.2 Immunofluorescence (IF)
2.3 Chromatin Immunoprecipitation Sequencing (ChIP-Seq)
2.4 TAM Single Cell ATAC Multiome ATAC + Gene Expression
3 Methods
3.1 Immunohistochemistry (IHC)
3.1.1 Tissue Fixation, Dehydration, and Paraffin Embedding
3.1.2 Tissue Section Slide Preparation
3.1.3 Heat-Induced Antigen Retrieval
3.1.4 Staining for Macrophage Marker F4/80 and NF-κB p65
3.2 Double Immunofluorescence (IF)
3.2.1 Slide Preparation
3.2.2 Double Immunofluorescence (IF) Staining
3.3 Chromatin Immunoprecipitation Sequencing (ChIP-Seq)
3.3.1 Tumor Tissue Cell Dissociation
3.3.2 Red Blood Cell Lysis
3.3.3 Dead Cell Removal
3.3.4 Macrophage Isolation
3.3.5 Chromatin Preparation and Immunoprecipitation
Cross Linking and Cell Lysis
Sonication of Isolated Chromatin to Shear DNA
Immunoprecipitation (IP) of Cross-Linked Protein/DNA
Elution of Protein/DNA Complexes and Protein/DNA Complex Crosslink Reversal
DNA Purification
Verify ChIP DNA Enrichment by Real-Time Quantitative PCR (RT-qPCR)
3.4 Single Cell Multiome ATAC + Gene Expression
3.4.1 Nuclei Isolation
3.4.2 Nuclei Transposition
3.4.3 GEM Generation and Barcoding
3.4.4 Post-GEM Cleanup
Post-GEM Incubation Cleanup: Dynabeads
Post-GEM Incubation Cleanup: SPRIselect
3.4.5 Pre-amplification PCR
Pre-amplification PCR
Post Pre-amplification PCR Cleanup
3.4.6 ATAC Library Construction
Sample Index PCR
Post Sample Index PCR Double-Sided Size Selection
3.4.7 cDNA Amplification
cDNA Amplification
cDNA Cleanup
3.4.8 Gene Expression Library Construction
Fragmentation, End Repair, and A-Tailing
Post Fragmentation, End Repair, and A-Tailing Double-Sided Size Selection
Adaptor Ligation
Post Ligation Cleanup
Sample Index PCR
Post Sample Index PCR Double-Sided Size Selection
3.4.9 Sequencing
4 Notes
References
Chapter 14: Methods to Study the Effect of IKK Inhibition on TNF-Inducing Apoptosis and Necroptosis in Cultured Cells
1 Introduction
2 Materials
2.1 Inducing Cell Death in Cultured Cell Lines
2.2 Evaluation of ;Cell Death by ATP Level and Caspase Assay
2.3 Co-immunoprecipitation and Immunoblotting
3 Methods
3.1 Cell Viability and Caspase Activity Detection
3.2 Isolation of FADD- and MLKL-Containing Complexes
3.2.1 Cell Stimulation and Detection of Phosphorylated MLKL (pSer345-MLKL)
3.2.2 Sequential Immunoprecipitation of FADD- and MLKL-Containing Complexes
4 Notes
References
Chapter 15: Use of ChIP-qPCR to Study the Crosstalk Between HIF and NF-κB Signaling in Hypoxia and Normoxia
1 Introduction
2 Materials
2.1 Cell Culture (See Note 1)
2.2 Treatments
2.3 ChIP Assay (See Note 2)
2.4 qPCR Analysis
3 Methods
3.1 Cell Culture Growth Conditions
3.2 Treatments
3.3 ChIP-qPCR
3.3.1 Formaldehyde Cross-Linking and Chromatin Extraction
3.3.2 Sonication
3.3.3 Preparing the Protein G-Sepharose Slurry
3.3.4 Lysate Pre-clearing
3.3.5 Immunoprecipitation (IP)
3.3.6 Immune Complex Capture, Washing, and Elution
3.3.7 Reverse Cross-Linking and DNA Purification
3.3.8 qPCR Analysis
4 Notes
References
Chapter 16: Methods to Analyze the Roles of TAK1, TRAF6, and NEMO in the Regulation of NF-κB Signaling by RANK Stimulation Dur...
1 Introduction
2 Materials
2.1 Isolation of Bone Marrow Macrophage Cells (BMMs) and Osteoclast Culture
2.2 Real-Time PCR Analyzing Osteoclast Differentiation
2.3 Western Blot for NF-κB Activation and Osteoclast Differentiation
2.4 NF-κB Luciferase Reporter Assay
2.5 Co-immunoprecipitation to Analyze the Interaction of Different Proteins Involved in NF-κB Signaling
2.6 Micro-CT Analysis
2.7 Histo-morphometric Analysis
2.8 Generation of Retroviral Particles to Study Role for TAK1 and NEMO Modulation
2.9 Immunofluorescence for ISGylated NEMO
3 Methods
3.1 RANKL Induced NF-κB Activation During Osteoclast Differentiation
3.1.1 Osteoclast Culture
Isolation of Bone Marrow Macrophage Cells (BMMs)
Differentiation of BMMs to Osteoclast (TRAP Staining)
Real-Time PCR for Analyzing Osteoclast Differentiation Markers
Western Blotting for NF-κB Activation and Osteoclastogenesis
NF-κB Luciferase Reporter Assay
3.2 Analyzing the Role of TAK1 During Osteoclastogenesis
3.2.1 In Vivo Effect of TAK1 Deletion on Osteoclastogenesis and Bone Parameters
Micro-CT Analysis for Bone Parameters (see Note 2)
Histo-morphometric Analysis
3.2.2 Ex Vivo Characterization of TAK1-Null BMMs for Osteoclastogenesis and NF-κB Activation
Retroviral Vector Mediated Transduction
Osteoclast Differentiation, mRNA Expression, and Western Blot Analysis
3.3 Analyzing Interaction of TRAF6 with NF-κB Components During Osteoclast Differentiation
3.3.1 Analyzing TRAF6-K63-Ubiquitnatoin
3.4 ISGylation of NEMO to Modulate NF-κB Activity
3.4.1 ISGylation of NEMO (Fig. 3)
3.4.2 Immuno-Colocalization of ISG15 and NEMO in Pre-osteoclast (Fig. 4)
3.4.3 Generation of Retroviral Particles and Osteoclastogenesis
4 Notes
References
Chapter 17: NF-κB Signaling in Ex-Vivo Mouse Intestinal Organoids
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Generation of Ex-Vivo Intestinal Organoids
3.2 Passage and Expansion of Ex-Vivo Intestinal Organoids
3.3 Treatment of Ex-Vivo Intestinal Organoids with TNFα
4 Notes
References
Chapter 18: Extracellular Flux Analysis to Investigate the Impact of NF-κB on Mitochondrial Respiration in Colorectal Carcinom...
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
3 Methods
3.1 Seahorse XFe96 Assay: Day 0
3.1.1 Seeding the Cells
3.1.2 Hydrating the XFe96 Sensor Cartridge
3.1.3 Designing the Experiments
3.2 Seahorse XFe96 Assay: Day 1
3.2.1 Preparing the Assay Medium and the Compound Working Solutions
3.2.2 Running the Assay
3.3 Data Analysis
4 Notes
References
Chapter 19: Conditional Knockout Mouse Models to Study the Roles of Individual NF-κB Transcription Factors in Lymphocytes
1 Introduction
1.1 Conditional Knockout NF-κB Subunit Alleles
2 Materials
2.1 PCR
2.2 Immunization with Sheep Red Blood Cells (SRBC)
2.3 Sample Generation from Mouse Tissues Following Immunization
2.4 Flow-Cytometric Analysis
2.5 Preparation of Tissue for Immunohistochemistry (IHC)
2.6 IHC of Lymphoid Tissue
2.7 Acquisition of Blood Samples and Serum Preparation
2.8 ELISA
3 Methods
3.1 PCR to Detect Cre-Mediated Deletion of Transgenic NF-κB Subunit Alleles
3.2 Primary and Secondary Immunization with SRBC
3.2.1 Preparation of SRBCs
3.2.2 Immunization
3.3 Sample Generation from Mouse Tissues Following Immunization for Flow Cytometry
3.4 Flow-Cytometric Analysis of Cell Subsets from Immunized Mice
3.5 Preparation of Tissue for IHC
3.6 IHC of Lymphoid Tissue
3.6.1 Deparaffinization
3.6.2 Antigen Retrieval
3.6.3 IHC
3.6.4 Counterstain and Mounting
3.7 Acquisition of Blood Samples and Serum Preparation
3.8 ELISA
4 Notes
References
Chapter 20: Generation and Surgical Analysis of Genetic Mouse Models to Study NF-κB-Driven Pathogenesis of Diffuse Large B Cel...
1 Introduction
2 Materials
2.1 Generating Conditional Mutations in B Cells
2.2 Detecting Lymphoma Development by Ultrasound
2.3 Partial Splenectomy with Recovery
2.4 Obtaining Tumoral Specimens by Dissection
3 Method
3.1 Generating Conditional Mutations in B Cells
3.2 Detecting Lymphoma Development by Ultrasound
3.3 Partial Splenectomy with Recovery
3.4 Obtaining Tumoral Specimens by Dissection
4 Notes
References
Chapter 21: The Screening of Combinatorial Peptide Libraries for Targeting Key Molecules or Protein-Protein Interactions in th...
1 Introduction
2 Materials
2.1 Protein Biotinylation
2.2 ELISA Competition Assay
3 Methods
3.1 Tetrapeptide Library Design and Synthesis
3.2 Protein Purification and Biotinylation
3.3 Iterative Deconvolution of the Combinatorial Tetrapeptide Library
3.3.1 ELISA Competition Assay
3.3.2 Synthesizing the Second-Generation Library
3.3.3 Examining the Dose-Dependent Effects
3.3.4 Synthesizing D-Enantiomers
3.3.5 Examining the Tetrapeptide Stability in Biological Fluids
3.4 Undertaking the Hit-To-Lead and Lead Optimization of the Selected D-Tetrapeptides
4 Notes
References
Index