دانلود کتاب Wnt Signaling: Methods and Protocols (Methods in Molecular Biology, 1481)

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Preface
Contents
Contributors
Chapter 1: Visualizing Wnt Palmitoylation in Single Cells
1 Introduction
2 Materials
2.1 Click Chemistry for Labeling Proteome Palmitoylation
2.2 Proximity Ligation Imaging Assay for Visualization of Palmitoylated Wnt3a
3 Methods
3.1 Click Chemistry for Labeling Proteome Palmitoylation
3.2 Proximity Ligation Imaging Assay for Visualizing Palmitoylated Wnt3a
3.3 Imaging Palmitoylated Wnt3a with a Confocal Fluorescence Microscope
4 Notes
References
Chapter 2: Monitoring Wnt Protein Acylation Using an In Vitro Cyclo-­Addition Reaction
1 Introduction
2 Materials
2.1 Cell Labeling Components
2.2 Protein Purification Reagents
2.3 Cycloaddition Reaction Reagents
2.4 SDS-PAGE and Immunoblotting Components
3 Methods
3.1 Labeling Wnt Protein
3.2 Purifying Wnt Protein
3.3 Azide Attack
3.4 SDS-PAGE and Immunoblotting
3.5 Detection
4 Notes
References
Chapter 3: Biochemical Methods to Analyze Wnt Protein Secretion
1 Introduction
2 Materials
2.1 Production of Wnt Conditioned Medium (CM)
2.2 Blue Sepharose Purification of Wnts
2.3 Purification of Extracellular Vesicles
2.4 Preparation of Control Lysate
2.5 Immunoblotting
2.6 Wnt/TCF4 Reporter Assay
3 Methods
3.1 Production of Wnt Conditioned Medium (CM)
3.2 Blue Sepharose Purification of Wnts
3.3 Purification of Extracellular Vesicles
3.4 Preparation of Control Lysate
3.5 Immunoblotting
3.6 Wnt/TCF4 Reporter Assay (Paracrine)
4 Notes
References
Chapter 4: Methods for Studying Wnt Protein Modifications/Inactivations by Extracellular Enzymes, Tiki and Notum
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Protein Purification
2.3 Metabolic Labeling of Wnt3a Acylation and Click Chemistry Reaction
3 Methods
3.1 In Vitro Cleavage of Wnt3a by TIKI2
3.2 Metabolic Labeling of Wnt3a Acylation
3.3 In Vitro Deacylation of Wnt3a by Notum
4 Notes
References
Chapter 5: Probing Wnt Receptor Turnover: A Critical Regulatory Point of Wnt Pathway
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Flow Cytometry
2.3 Surface Biotinylation Reagents
2.4 SDS-PAGE and Western Blot
2.5 SNAP-tag, SNAP-tag Probe
3 Methods
3.1 Flow Cytometry to Detect Cell Surface Wnt Receptors FZD and LRP6
3.2 FZD Ubiquitination
3.2.1 Surface Labeling and Lysis
3.2.2 NeutrAvidin Pull-down
3.2.3 Myc Pull-down
3.3 Surface FZD Labeling in Live Cells
4 Notes
References
Chapter 6: A Simple Method to Assess Abundance of the β-Catenin Signaling Pool in Cells
1 Introduction
2 Materials
2.1 Equipment and Disposables
2.2 Reagents for GST-ICAT Fusion Protein Purification
2.3 Reagents for GST-ICAT Pull-Down
2.4 Reagents for Western Blot
3 Methods
3.1 Transforming Protein-­Competent Bacterial Cells with GST-ICAT
3.2 Purification of GST-ICAT Fusion Protein
3.3 Lysing Cells
3.4 Preparing Glutathione-
3.5 GST-ICAT Pull-Down
3.6 Western Blot for β-Catenin
4 Notes
References
Chapter 7: Wnt-Dependent Control of Cell Polarity in Cultured Cells
1 Introduction
2 Materials
2.1 Measuring Wnt5a-Induced Polarization of Cells
2.2 Live Cell Imaging of Wnt5a-­Induced Polarity
2.3 Wnt5a-Mediated Polarization in Response to a Directional Cue
3 Methods
3.1 Measuring Wnt5a-Induced Polarization of Cells
3.2 Live Cell Imaging of Wnt5a-­Induced Polarity
3.3 Wnt5a-Mediated Polarization in Response to a Directional Cue
4 Notes
References
Chapter 8: The Use of Chick Embryos to Study Wnt Activity Gradients
1 Introduction
2 Materials
2.1 Electroporation
2.2 Embedding and Sectioning
2.3 Immunostaining
3 Methods
3.1 Incubating Eggs
3.2 Loading Needles
3.3 Windowing Eggs
3.4 Injecting and Electro­porating
3.5 Harvesting and Embedding Embryos
3.6 Immunostaining Embryos
3.7 Imaging Embryos
4 Notes
References
Chapter 9: Monitoring Wnt Signaling in Zebrafish Using Fluorescent Biosensors
1 Introduction
2 Materials
2.1 Generation of Transgenic Lines: Cloning and Microinjection
2.2 Zebrafish Embryo Medium Preparation
2.3 In Vivo Embryo Treatment
2.4 Image Analysis and Statistics
3 Methods
3.1 Generation of Wnt-­Responsive Vectors Using the MultiSite Gateway System
3.2 In Vitro Transcription of Capped Tol2 Transposase mRNA
3.3 Injection of Tol2 Plasmid and Transposase mRNA into Zebrafish Embryos
3.4 Generation of Stable Wnt-Reporter Zebrafish Lines
3.5 Pharmacological Validation of Tissue-­Specific Wnt Reporter Fish
3.6 Digital Quantification of Integrated Density Using Fiji Image Processing Software
4 Notes
References
Chapter 10: Biochemical Analysis of Tankyrase Activity in Zebrafish In Vitro and In Vivo
1 Introduction
2 Materials
2.1 Derivation of a Cell Culture from Zebrafish Embryos
2.2 Drug Treatment and Tissue Collection from Zebrafish Adult Fish
2.3 Western Blotting of Zebrafish Cell Cultures and Adult Tissues
3 Methods
3.1 Derivation of Embryonic Cell Culture from Zebrafish Embryos
3.2 Drug Treatment and Tissue Collection from Zebrafish Adult Fish
3.3 Western Blotting of Zebrafish Cell Cultures and Adult Tissues
4 Notes
References
Chapter 11: Reconstitution of the Cytoplasmic Regulation of the Wnt Signaling Pathway Using Xenopus Egg Extracts
1 Introduction
1.1 The Wnt Signaling Pathway
1.2 Use of Xenopus Egg Extracts to Study the Wnt Signaling Pathway
1.3 Xenopus Egg Extract as a Tool for Drug Discovery
2 Materials
3 Methods
3.1 Harvesting Xenopus Eggs
3.1.1 Inject Female Xenopus laevis
3.1.2 Harvest Xenopus Eggs
3.2 Preparation of Xenopus Egg Extract
3.2.1 Centrifugation of Xenopus Eggs
3.2.2 Collect and Prepare the Cytoplasmic Layer of Extract
3.3 Depletion of Extracts
3.3.1 Deplete Xenopus Egg Extracts of Endogenous Axin
3.4 Degradation Assay
3.4.1 Prepare Radiolabeled β-catenin
3.4.2 Prepare Egg Xenopus Extract for Degradation Assay
3.4.3 Perform Degradation Assay
3.4.4 Perform β-Catenin Luciferase Degradation Assay
4 Notes
References
Chapter 12: Delivery of the Porcupine Inhibitor WNT974 in Mice
1 Introduction
2 Materials
2.1 Drug Preparation
2.2 Drug Delivery
3 Methods
3.1 Drug Preparation
3.2 Drug Delivery
4 Notes
References
Chapter 13: Use of Primary Calvarial Osteoblasts to Evaluate the Function of Wnt Signaling in Osteogenesis
1 Introduction
2 Materials
2.1 Calvarial Cell Isolation
2.2 Gene Knockout with Adenoviral Infection
2.3 Osteogenic Differentiation
2.4 Alkaline Phosphatase Quantification
2.5 Alizarin Red Staining
2.6 Von Kossa Staining
3 Methods
3.1 Calvarial Cell Isolation
3.2 Gene Knockout with Adenoviral Infection
3.3 Osteogenic Differentiation
3.4 Alkaline Phosphatase Quantification
3.5 Alizarin Red Staining
3.6 Von Kossa Staining
4 Notes
References
Chapter 14: Monitoring Wnt/β-Catenin Signaling in Skin
1 Introduction
2 Materials
2.1 Wnt Reporter Mice
2.2 Tissue Embedding Components
2.3 Tissue Fixation Reagents and Components
2.4 Tissue Sectioning and Staining Components
2.5 Primary and Secondary Antibodies
2.6 X-gal Staining
2.7 Microscopy and Imaging Setup
3 Methods
3.1 Harvesting Backskin Tissue
3.2 Prefixation of Tissue for GFP Preservation
3.3 Embedding Backskin in OCT Block
3.4 Cryosectioning
3.5 Visualizing GFP Signal in GFP Reporter Mouse Skin
3.6 X-gal Staining of lacZ Reporter Mouse Skin
3.7 Immunostaining for Nuclear β-catenin
3.7.1 Dehydration and Antigen Retrieval of FFPE Sample
3.7.2 Blocking and Antibody Staining with M.O.M Kit
3.7.3 Signal Amplification and Development
3.7.4 Hematoxylin Counterstain and Imaging
4 Notes
References
Chapter 15: The Generation of Organoids for Studying Wnt Signaling
1 Introduction
2 Materials
2.1 Generation and Maintenance of Organoid Cultures Derived from Human Intestinal Tissue
2.1.1 Isolation of Crypts from Human Intestinal Tissue
2.1.2 Culturing of Human Intestinal Crypts
2.1.3 Passaging of Human Intestinal Organoids
2.2 Lentiviral Transduction of Mouse and Human Intestinal Organoids
2.2.1 Production of Lentiviruses
2.2.2 Lentiviral Transduction of Intestinal Organoids
2.3 CRISPR/Cas9-mediated Genome Editing in Mouse and Human Intestinal Organoids
2.3.1 Design and Cloning of the Cas9 and sgRNA Expression Construct
2.3.2 Bacteria Transformation with sgRNA Constructs
2.3.3 Plasmid Purification and Sequencing
2.3.4 Preparation of Lipofection Mixes
2.3.5 Preparation and Lipofection of Mouse and Human Intestinal Organoids
2.3.6 Genotyping Organoids: Genomic DNA Extraction from Organoid Culture
2.3.7 Genotyping Organoids: PCR Amplification of the Targeted Region
2.3.8 Genotyping Organoids: Cloning of the PCR Product and Sequencing of the Cloned Fragment
3 Methods
3.1 Generation and Maintenance of Organoid Cultures Derived from Human Intestinal Tissue
3.1.1 Isolation of Crypts from Human Intestinal Tissue
3.1.2 Culturing of Human Intestinal Crypts
3.1.3 Passaging of Human Intestinal Organoids
3.2 Lentiviral Transduction of Intestinal Organoids
3.2.1 Production of Lentiviruses for the Transduction of Intestinal Organoids
3.2.2 Lentiviral Transduction of Mouse and Human Intestinal Organoids
3.3 CRISPR/Cas9-mediated Genome Editing in Mouse and Human Intestinal Organoids
3.3.1 Design and Cloning of the Cas9 and sgRNA Expression Construct
3.3.2 Bacteria Transformation with sgRNA Constructs
3.3.3 Plasmid Purification and Sequencing
3.3.4 Preparation of Lipofection Mixes
3.3.5 Preparation and Lipofection of Mouse and Human Intestinal Organoids
3.3.6 Genotyping Organoids: Genomic DNA Extraction from Organoid Culture
3.3.7 Genotyping Organoids: PCR Amplification of the Targeted Region
3.3.8 Genotyping Organoids: Cloning of the PCR Product and Sequencing of the Cloned Fragment
4 Notes
References
Chapter 16: Methods to Manipulate and Monitor Wnt Signaling in Human Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents, Stock Solutions and Consumables
3 Methods
3.1 Passaging hPSCs
3.2 Manipulating WNT Signaling in hPSCs
3.3 Detecting Wnt Activity
3.3.1 Morphology Based Detection by Light Microscopy
3.3.2 Gene Expression Based Detection by qPCR
3.3.3 Reporter Based Detection with Flow Cytometry
4 Notes
References
Chapter 17: Directed Endothelial Progenitor Differentiation from Human Pluripotent Stem Cells Via Wnt Activation Under Defined Conditions
1 Introduction
2 Materials
2.1 Cell Culture and Differentiation Reagents
2.2 Magnetic-
2.3 Characterization of hPSC-derived Endothelial Cells
2.4 Cryostorage and Thawing of Cells
3 Methods
3.1 Endothelial Progenitor Differentiation with Gsk3 Inhibitor
3.2 Purification of Bipotent Endothelial Progenitors
3.3 Extended Culture of hPSC-derived Endothelial Progenitors to Endothelial Cells
3.4 Characterization of hPSC-derived Endothelial Cells and Their Progenitors
3.5 Cryostorage and Thawing of Endothelial Cells and Their Progenitors
4 Notes
References
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Index