Chemokines, Part A

Cover Page
......Page 1
Series Editor
......Page 2
Copyright page
......Page 3
Contributors
......Page 4
Preface
......Page 12
Methods in Enzymology
......Page 14
Chemokines in Human Breast Tumor Cells: Modifying Their Expression Levels and Determining Their Effects on the Malignancy Phenotype......Page 41
Introduction......Page 42
Transfection (microporation) procedures: MCF-7, T47D, and MDA-MB-231 cells......Page 43
Additional materials......Page 44
On the day of microporation......Page 45
Tumor cell handling after microporation......Page 46
Establishment of Primary Local Breast Tumors and Pulmonary Metastases......Page 47
Formation of primary tumors by T47D cells......Page 48
One day prior to tumor cell injection......Page 49
Mice and supplementary material for tumor cell injection......Page 50
Disposables:......Page 51
Determining formation of pulmonary metastases......Page 52
References......Page 53
CCR5 Pharmacology Methodologies and Associated Applications
......Page 55
CXCR4 and Mobilization of Hematopoietic Precursors......Page 94
Introduction......Page 56
Protein Expression......Page 57
Materials......Page 292
CXCL12 preparation......Page 358
Example data and results......Page 61
FACS Assay......Page 62
Flow cytometric enumeration of murine HSPCs......Page 63
Dosing and sampling regimen......Page 65
GTP-associated CCR5 inverse agonism assay......Page 66
FACS-based chemokine uptake assays......Page 281
cAMP-response-element-luciferase reporter gene assay......Page 68
Transient transfections and assay plate preparation......Page 69
Example results/data......Page 70
Radiolabeled CCR5 chemokine-binding assays......Page 71
Immobilization of 1D4 monoclonal antibody on a CM4 surface......Page 72
Real time HIV-1 gp120-CCR5 binding assay......Page 73
Inhibition of soluble recombinant HIV-1 gp120 (Ba-L strain) binding to CCR5 by TRFIA......Page 74
Application of gp120 binding to characterize functional occupancy in vitro......Page 75
HIV-1 gp160-CCR5-mediated cell-cell fusion assay......Page 76
Cell preparation: Peripheral blood lymphocytes......Page 78
Reverse transcriptase assay......Page 79
HIV‐1 resistance to CCR5 antagonists......Page 80
CCR5 Site-Directed Mutagenesis and Ligand Docking Studies......Page 81
Structural model generation......Page 82
Transfection of HEK Gα15 cells with pIRESneo-CCR5 (various isoforms) and pCRE-luc......Page 83
Example results/data......Page 84
Vector construction for hCCR5 knock-in......Page 86
Transfection and human CCR5 knock-in mouse generation......Page 87
Materials......Page 88
References......Page 236
Introduction......Page 95
HSPC Mobilizing Agents that Target the CXCL12/CXCR4 Axis......Page 96
TB infection model......Page 101
Preparation of media from virus-infected cell cultures......Page 207
Flow cytometric enumeration of human HSPCs......Page 102
Mobilization of human HSPCs by G-CSF......Page 103
Mobilization of human HSPCs by plerixafor......Page 104
Flow Cytometric Analysis of CXCR4 Expression on Human CD34+ Subsets......Page 106
Evaluation of cell surface CXCR4 on human CD34+ cell subsets......Page 107
Transmigration assays......Page 110
Viral production......Page 165
Transplantation assays for mouse and human HSPCs......Page 111
Limiting dilution competitive repopulation assay......Page 112
Immune-deficient mouse models to study human stem cell-repopulation capacity......Page 114
References......Page 117
References......Page 221
Double-Label Nonradioactive in Situ Hybridization for the Analysis of Chemokine Receptor Expression in the Central Nervous System......Page 128
Introduction......Page 129
Equipment and reagent......Page 130
Total RNA purification......Page 131
Scintillation-proximity assay......Page 132
Generation of ISH probe by in vitro transcription......Page 133
Chemotaxis assay in micro fluidic gradient device......Page 134
Development of ISH signals......Page 135
Double ISH......Page 136
Fluorescent double ISH......Page 137
Comments......Page 138
References......Page 139
Expression of Chemokines and Chemokine Receptors in Human Colon Cancer......Page 141
Introduction......Page 142
TaqMan Low Density Array......Page 144
Chemokine neutralization in vivo......Page 145
TaqMan Low Density Array analysis of eight colon cancer samples......Page 146
CCL3, CCL4, and CXCL8 expression in colon cancer......Page 148
Regulation of CXCL8 expression in colon cancer cell lines......Page 151
CXCL8 correlation with OPN and SPARC......Page 152
vGPCR Transforming Activity In Vivo Using Xenograft Systems......Page 153
References......Page 155

Kaposi's Sarcoma Virally Encoded, G-Protein-Coupled Receptor: A Paradigm for Paracrine Transformatio......Page 158
Introduction......Page 160
Cloning of vGPCR......Page 161
Cell lines, plasmids, and transfection......Page 274
Procedure......Page 162
Overview......Page 163
FlashPlate assay......Page 214
Overview......Page 164
Overview......Page 167
Outline......Page 168
Procedure......Page 169
Procedure......Page 170
Overview......Page 171
Experiment......Page 172
Procedure......Page 173
Overview......Page 174
Procedure......Page 175
Overview......Page 176
Overview......Page 177
Procedure......Page 178
Procedure......Page 179
References......Page 180
Pharmacological and Biochemical Characterization of Human Cytomegalovirus-Encoded G Protein-Coupled Receptors......Page 184
Introduction......Page 185
Virally Encoded GPCR Engineering......Page 187
Enzyme-linked immunosorbent assay......Page 189
Radioligand binding assays......Page 190
Inositol phosphate production......Page 192
Reporter gene assays......Page 193
Cellular transformation: Foci formation assay......Page 195
Cell proliferation assay: Cyclin D1 expression......Page 196
In vivo xenograft models......Page 197
Generation of Recombinant HCMV Strains by Markerless Bacterial Artificial Chromosome Mutagenesis......Page 198
References......Page 200
Identification and Characterization of Virus-Encoded Chemokine Binding Proteins
......Page 205
Introduction......Page 206
M-T7 inhibits inflammation and vasculopathic disease in animal models......Page 210
Cell transfection......Page 211
Surface plasmon resonance to characterize vCKBP-chemokine interactions......Page 216
The use of SPR in GAG competition assays......Page 219
SPR technology to investigate the interaction between vCKBPs and GAGs......Page 220
The Chemokine-Binding Protein M3 as a Tool to Understand the Chemokine Network 
......Page 224
Introduction......Page 316
Generation of Transgenic Mice Expressing M3 in Insulin-Producing beta Cells......Page 226
M3 Expression in Islets of Langerhans Blocks CCL2-, CCL21-, and CXCL13-Induced Migration of Cells to Isle......Page 229
Radioactive Phosphorylation of CXCR2 Interacting Proteins......Page 349
Generation of a Conditional Transgenic System for Expression of M3......Page 233
Concluding Remarks......Page 235
M-T7: Measuring Chemokine-Modulating Activity
......Page 239
Chemokine-glycosaminoglycan interaction......Page 240
Discovery and identification of the M-T7 gene......Page 241
Generation of viral constructs......Page 243
Reagents......Page 301
Cell adhesion......Page 246
Membrane fluidity......Page 247
Ascites assay......Page 249
Aortic transplant model......Page 250
Aortic transplant recipient......Page 251
Tissue staining......Page 253
Morphometric analysis of aortic plaque area......Page 254
Preclinical Toxicity Testing......Page 255
References......Page 269
Role of the Chemokine Scavenger Receptor D6 in Balancing Inflammation and Immune Activation
......Page 259
Immunohistochemistry......Page 260
Chemokine scavenging assay......Page 262
Results......Page 263
Discussion......Page 264
Structure-Function Dissection of D6, an Atypical Scavenger Receptor......Page 272
Introduction......Page 273
GST-CXCR2 C-tail constructs......Page 346
Biotinylated chemokines......Page 277
Scavenging assays......Page 278
Tracking atypical receptors with antibodies......Page 279
Using antibodies to study receptor recycling......Page 280
D6 as a model for determining chemokine receptor structure......Page 282
Generation of heterologous transfectants expressing D6......Page 283
Bioreactor protocol......Page 284
Solubilization using DDM......Page 285
Functional assays......Page 286
References......Page 287
Modeling Small Molecule-Compound Binding to G-Protein-Coupled Receptors
......Page 289
Similarity and Differences in the Crystal Structures of Class-A GPCRs Solved to Date......Page 291
Ab Initio modeling methods......Page 293
Method......Page 294
Ligand docking to ab initio model of CCR1......Page 296
Computational Methods for Receptor Flexibility and Ligand-Induced Conformational Changes in GPCRs......Page 297
Liticon method......Page 299
Peripheral Spying of Intratumoral Signaling of Conscious Mice......Page 377
Method......Page 302
Method......Page 303
Chemotaxis and chemokinesis assays in modified Boyden chamber......Page 326
Stock solution......Page 304
Method......Page 305
Method......Page 307
Method......Page 308
Conclusions......Page 310
References......Page 312
Elucidation of Chemerin and Chemokine-Like Receptor-1 Function in Adipocytes by Adenoviral-Mediated Shrna knowckdown of Gne Expression
......Page 315
The 3T3-L1 Cell Model for Adipogenesis and Adipocyte Metabolism......Page 318
RNA Interference......Page 319
Titration of adenoviral shRNA particles......Page 320
Adipocyte differentiation media......Page 323
Testing the efficacy of CE- and CR-shRNA adenoviral vectors......Page 324
Maintenance and preparation of 3T3-L1 cells......Page 325
RNA isolation and quantification of chemerin and CMKLR1 knock-down by quantitative PCR......Page 328
Postdifferentiation knock-down of chemerin and CMKLR1......Page 330
Effect of chemerin and CMKLR1 knock-down on adipogenesis (oil red O staining)......Page 332
Effect of chemerin and CMKLR1 knock-down on adipocyte metabolism......Page 333
Acknowledgments......Page 335
References......Page 398
Characterization of Chemokine Receptor CXCR2 Interacting Proteins Using a Proteomics Approach to Define the CXCR2 "Chemosynapse"
......Page 339
Introduction......Page 340
The CXCR2 chemosynapse......Page 341
Proteomic screen for the CXCR2 chemosynapse adaptor proteins......Page 342
Validation of the Interaction of Novel Proteins with CXCR2......Page 344
Polarization in Zigmond chamber......Page 345
Production of GST-fusion proteins......Page 347
Mutational Analysis of Residues at Interactive Interface of CXCR2 and CXCR2-Binding Proteins......Page 348
Chemotaxis Assay......Page 350
References......Page 353
Phosphoproteomic Analysis of Chemokine Signaling Networks......Page 355
Introduction......Page 356
Denaturation, reduction, and alkylation......Page 359
Trypsin digest......Page 360
IMAC bead preparation and enrichment......Page 361
Additional phosphoenrichment strategies and considerations......Page 362
Reversed-phase liquid chromatography and tandem mass spectrometry......Page 363
Phosphopeptide identification with InsPecT......Page 364
Considerations for selecting an appropriate search database program......Page 366
Summary......Page 367
Acknowledgments......Page 368
References......Page 369
Monitoring NF-kappaB Mediated Chemokine Transcription in Tumorigenesis......Page 371
Development of NF-kappaB Reporter Model for Tumors......Page 372
Gaussian luciferase reporter......Page 373
Cell-Based Assays for Kinase and Transcriptional Activity In Vitro......Page 376
References......Page 378
Analysis of Chemokine Receptor Endocytosis and Intracellular Trafficking......Page 380
Introduction......Page 381
Receptor Detection......Page 383
Cells......Page 385
Flow cytometry......Page 386
Pre-labeling of cell-surface receptors......Page 387
Endocytosis of cell-surface receptors......Page 388
Monitoring Receptor Recycling......Page 389
Immunofluorescence microscopy......Page 390
Monitoring Receptor Degradation......Page 391
Western blotting......Page 392
Immunofluorescence......Page 393
Cell surface replicas of whole-mount preparations......Page 394
Preparation of membrane sheets......Page 395
Immuno-gold labeling of ultrathin cryosections......Page 397
Measuring the Proximity of T-Lymphocyte CXCR4 and TCR by Fluorescence Resonance Energy Transfer (FRET)
......Page 401
Introduction......Page 402
What is FRET?......Page 403
Advantages of PE/APC mAb FRET......Page 404
CFP/YFP fusion protein FRET......Page 406
Using both FRET approaches to study CXCR4-TCR proximity......Page 407
Important considerations for labeling cell-surface CXCR4 and TCR......Page 408
Detailed procedure......Page 409
Examples and results......Page 410
Jurkat results and controls......Page 411
Plasmids and controls......Page 414
Detailed protocol for transient transfection......Page 415
Preparation of transfected cell samples......Page 416
Analysis and interpretation of fluorescent emission spectra......Page 417
References......Page 418
Expression of CXCR4, a G-Protein-Coupled Receptor for CXCL12 in Yeast: Identification of New&h......Page 420
Introduction......Page 421
Overview of yeast-signaling strategies......Page 423
Experimental approach......Page 424
Characterization of inverse agonists for CXCR4......Page 425
Summary......Page 429
References......Page 431
Ubiquitination of Chemokine Receptors......Page 434
Introduction......Page 435
Cell Culture and Transfections......Page 437
Agonist Treatment and Ubiquitination Assay......Page 438
E3 Ubiquitin Ligase AIP4 Mediates Ubiquitination of CXCR4......Page 440
References......Page 442
Author Index
......Page 444
Subject Index
......Page 472