The unfolded protein response and cellular stress, Part 3

CFTR Expression Regulation by the Unfolded Protein Response
......Page 38
Introduction......Page 39
ER stress induction......Page 41
RNA isolation......Page 42
Semiquantitative RT-PCR using ABI 7500 Real-Time PCR System
......Page 43
Assessment of UPR reporter (spliced XBP1 and BiP/HSPA5) mRNA levels by semiquantitative RT-PCR
......Page 44
Determination of CFTR mRNA stability
......Page 45
Assessment of cytoplasmic RNase activity
......Page 46
CFTR mRNA half-life
......Page 47
CFTR promoter reporter assay
......Page 48
Chromatin immunoprecipitation assays
......Page 50
Assessment of CFTR protein levels
......Page 53
CFTR maturation efficiency measurements
......Page 54
Detection of steady-state CFTR levels by Western blot
......Page 55
References......Page 57
GRP78/BiP Chapter : Modulation of GRP78/BiP in Altering Sensitivity to Chemotherapy
......Page 60
Introduction......Page 61
Glioma cell lines and primary cell culture
......Page 62
Glioma tissue......Page 63
Immunohistochemistry
......Page 64
Meaurement of intermediate ER stress......Page 65
Modulation of GRP78/BiP levels in vitro......Page 66
Cell death assays......Page 67
EGCG modulation of GRP78......Page 68
Rodent glioma models-Subcutaneous and intracranial
......Page 69
Tissue analysis of GRP78 levels......Page 70
References......Page 71
Targeting the Unfolded Protein Response in Cancer Therapy
......Page 72
The UPR and Cancer......Page 73
Targeting the UPR in cancer cells via selective cleavage of GRP78
......Page 75
Viability of EGFR-positive cancer cells exposed to EGF-SubA
......Page 77
Required materials
......Page 78
Assessment of cell susceptibility to EGF-SubA
......Page 79
Analysis of selective GRP78 cleavage in cancer cells
......Page 80
Required materials
......Page 81
Tumor cell exposure and Western blot analysis
......Page 83
Combination of EGF-SubA with thapsigargin for treatment of MDA231luc cells
......Page 85
Combination of EGF-SubA with histone deacetylase inhibitors in the glioblastoma cell line T98G
......Page 87
Required materials......Page 88
References......Page 89
Large-Scale Analysis of UPR-Mediated Apoptosis in Human Cells
......Page 92
Introduction......Page 93
Monitoring Proliferation and Caspase Activation Following UPR Activation
......Page 95
Monitoring the Expression of UPR and Cell Death Target Genes
......Page 96
Cell culture......Page 97
Reverse transcription......Page 98
Quantitative reverse transcription real-time PCR
......Page 99
Semiquantitative PCR analysis of XBP1u and XBP1s with Cells-to-CT-derived cDNA
......Page 101
DNA Fragmentation Analysis......Page 103
References......Page 104
Quantitative Analysis of Amino Acid Oxidation Markers by Tandem Mass Spectrometry
......Page 107
Introduction......Page 109
Isotope dilution MS is a highly sensitive and specific method for detecting oxidized biomolecules in vivo
......Page 110
Oxidized amino acid content correlates with degree of oxidative stress in vivo
......Page 111
Buffers and solutions required for sample preparation
......Page 112
Protein precipitation, delipidation, and hydrolysis of lysates derived from biological samples
......Page 113
Preparation of authentic and isotope-labeled standards
......Page 114
Gas chromatography/Mass spectrometry (GC/MS)
......Page 115
LC/MS/MS detection of oxidized amino acids using authentic standards
......Page 116
Quantitation of oxidized amino acids by MRM analysis
......Page 118
Acknowledgments......Page 120
References......Page 121
Animal Models in the Study of the Unfolded Protein Response
......Page 124
Introduction......Page 125
Activating Transcription Factor 6......Page 126
IRE1/X-Box-Binding Protein-1......Page 127
PKR-Like ER Kinase......Page 130
eIF2α......Page 131
ATF4......Page 132
CHOP......Page 133
GADD34......Page 134
Transgenic Mouse Models for Monitoring ER Stress......Page 135
UPR and Lipid Metabolism......Page 136
UPR, Hypoxia, and Cancer......Page 137
Future Challenges......Page 138
References......Page 139
Measurement of Fluoride-Induced Endoplasmic Reticulum Stress Using Gaussia Luciferase
......Page 143
Introduction......Page 144
Properties of Gaussia Luciferase......Page 146
Cells......Page 147
Gluc lentivirus vector......Page 148
Luminometer......Page 149
Transduction (or transfection) with Gluc......Page 150
Measurement of Gluc activity......Page 152
Fluoride and Gluc activity......Page 153
Conclusions......Page 155
References......Page 156
Analysis of Nelfinavir-Induced Endoplasmic Reticulum Stress
......Page 158
Introduction......Page 159
Using Nelfinavir......Page 160
Fluorescent antibodies
......Page 161
Fluorescent proteins......Page 163
Fluorescent glibenclamide......Page 164
Immunoblot Analysis......Page 165
RT-PCR Analysis......Page 166
RNA preparation......Page 169
PCR and PCR conditions......Page 170
Conclusions......Page 172
References......Page 173
Using Temporal Genetic Switches to Synchronize the Unfolded Protein Response in Cell Populations In Vivo
......Page 174
Introduction......Page 175
Variable Functions of CHOP in the PERK Signaling Pathway
......Page 177
Mutations in the PLP1 Gene: A Model UPR Disease
......Page 178
Problems with Current Paradigms Used to Characterize the UPR
......Page 180
Required materials......Page 181
Construct design......Page 183
Part 2: A transgene construct to induce Cre recombinase expression in oligodendrocytes and trigger the genetic switch
......Page 184
Detection of oligodendrocytes in which the genetic switch has been triggered
......Page 185
Alternative knockin constructs for simultaneous UPR induction and labeling of oligodendrocytes
......Page 186
Applications of GST to the study of UPR diseases and neurodegeneration
......Page 187
Acknowledgments......Page 188
References......Page 189
Introduction......Page 193
Required materials......Page 195
Extraction and purification of Dol and Dol-P
......Page 196
Anthryldiazomethane labeling of Dol-P
......Page 197
Negative ion electrospray mass spectrometry of Dol-P
......Page 198
Required materials......Page 199
Hydrolysis and purification of oligosaccharides
......Page 200
HPLC analysis of 2AB-labeled oligosaccharides
......Page 201
N-glycosylation site occupancy......Page 202
Proteolytic digestion and deglycosylation of glycopeptides
......Page 203
Calculation of the N-glycosylation site occupancies
......Page 204
O-glycosylation......Page 205
Reductive beta-elimination......Page 206
MALDI-MS......Page 208
Hydrazinolysis......Page 209
References......Page 211
Monitoring and Manipulating Mammalian Unfolded Protein Response
......Page 213
Introduction......Page 214
Xbp-1 splicing assay for mammalian cells and tissues
......Page 215
Western blotting of eIF2α and ATF4......Page 217
Alternative approaches to UPR evaluation......Page 218
Chemical-Genetic Manipulation of Mammalian UPR......Page 219
Activation of IRE1[I642G]
......Page 220
Activation of wild-type ATF6
......Page 223
Concluding Remarks......Page 224
References......Page 225
A Screen for Mutants Requiring Activation of the Unfolded Protein Response for Viability
......Page 229
Introduction......Page 230
Primary screen......Page 232
Protocol for pulse-chase immunoprecipitation assay......Page 236
Pulse-chase......Page 237
Immunoprecipitation assay......Page 238
Cloning by complementation......Page 239
Library transformation and plasmid isolation......Page 240
Identification of mutant genes......Page 241
The beta-galactosidase assay......Page 242
SGA Database......Page 243
Closing Remarks......Page 244
References......Page 245
Signaling Pathways of Proteostasis Network Unrevealed by Proteomic Approaches on the Understanding of Misfolded Protein Rescue
......Page 247
Introduction......Page 248
2DE-Based Proteomics Approach Analysis to Study Protein Expression Profile
......Page 249
Low-temperature treatment and in vivo labeling
......Page 250
2D-PAGE......Page 251
Coomassie and fluorography......Page 252
Protein Identification by MS......Page 253
MALDI analysis-Equipment and specifications
......Page 254
Biochemical Validation......Page 255
Bioinformatics analysis......Page 256
Clustering analysis......Page 258
References......Page 261
Decreased Secretion and Unfolded Protein Response Upregulation
......Page 264
Introduction......Page 265
Heterologous Protein Expression......Page 266
Quality Control Mechanisms of the Secretory Pathway......Page 267
Experimental Systems to Evaluate Expression, UPR, and Secretory Processing
......Page 268
S. cerevisiae Strains Used for Optimal Expression......Page 269
scFv 4-4-20 construct......Page 270
UPR sensors......Page 271
Strain growth, expression, and isolation of intracellular heterologous protein
......Page 272
Analysis of GFP fusions by in-gel fluorescence
......Page 274
Experimental results......Page 275
Pulse-chase and immunoprecipitation protocol
......Page 276
Statistical Analysis of Microarray Results
......Page 277
Appropriate controls......Page 278
Selecting differentially expressed genes......Page 280
Classification......Page 281
Enrichment of promoter regions in known transcription factor-binding sites
......Page 282
Relative quantification by real-time quantitative PCR
......Page 283
Implications for protein secretion and UPR induction
......Page 285
References......Page 286
Measuring Signaling by the Unfolded Protein Response
......Page 290
Introduction......Page 291
Measuring Signaling by the UPR in S. cerevisiae
......Page 295
General guidelines for work with RNA......Page 297
Reagents......Page 298
Disposables......Page 299
Isolation of total RNA from S. cerevisiae......Page 300
Gel preparation......Page 301
Capillary transfer of RNA......Page 302
Random-primed labeling of DNA probes
......Page 303
Hybridization......Page 304
Measuring Signaling by the UPR in Mammalian Cells
......Page 305
Disposables
......Page 306
Extraction of RNA......Page 307
XBP1 splicing PCR assay
......Page 308
Protein methods for analyzing the UPR......Page 310
Required materials
......Page 313
Preparation of lysates
......Page 314
SDS-PAGE and immunoblotting
......Page 315
Acknowledgments......Page 317
References......Page 318
Quantitative Measurement of Events in the Mammalian Unfolded Protein Response
......Page 322
Introduction......Page 323
Activation of the UPR with ER stress inducers......Page 324
Northern Blots for GRP78/BiP and EDEM......Page 325
RT-PCR analysis of XBP1 mRNA splicing......Page 328
Gel condition determination......Page 329
Measurement with L-[35S] methionine
......Page 331
Radioactivity measurement with HPLC
......Page 332
Calculations of Transcript Accumulation, Signal Activation, and Correlation
......Page 333
Conclusion......Page 334
References......Page 335
Regulation of Immunoglobulin Synthesis, Modification, and Trafficking by the Unfolded Protein Response: A Quantitative Approach: A Quantitative Approach 
......Page 338
Introduction......Page 339
Isolation splenic B cells by magnetic sorting
......Page 341
Transfection of packaging cells
......Page 343
Retroviral transduction of B cells
......Page 344
Metabolic labeling of B cells
......Page 345
Cell lysis and immunoprecipitation
......Page 346
Measurement of Ig Mislocalization in Primary B Cells
......Page 349
Permeabilization of primary B cells using digitonin and trypsin digestion of cytoplasmic proteins
......Page 351
References......Page 353
Use of Chemical Genomics in Assessment of the UPR......Page 355
Introduction......Page 356
Assessment of the Activation of UPR Transcriptional Program in Cancer Cell
......Page 357
UPR-modulating compounds
......Page 358
Statistical analysis......Page 359
Identification of the glucose deprivation signature
......Page 360
The effect of UPR modulators on the glucose deprivation signature
......Page 361
Gene expression-based screening......Page 362
Transfection......Page 363
The effect of UPR modulators on the UPR signal pathway
......Page 364
Future Perspective of Chemical Genomics in UPR Research......Page 366
References......Page 367
Small GTPase Signaling and the Unfolded Protein Response
......Page 370
Introduction......Page 371
Materials and Methods......Page 373
Splicing of XBP1 mRNA......Page 375
Recombinant strategies......Page 377
GFP reporter assays in C. elegans
......Page 378
Immunofluorescence-based visualization of actin remodeling upon ER stress
......Page 380
GTPase pull-down assay......Page 382
Genetic modulation by RNA interference......Page 383
References......Page 385
Inhibitors of Advanced Glycation and Endoplasmic Reticulum Stress
......Page 388
Introduction......Page 389
Advanced Glycation and Its Pathophysiology
......Page 390
HPLC and GC/MS......Page 391
Immunohistochemistry......Page 392
IHC for the detection of CEL in the kidney with ischemia-reperfusion
......Page 393
Western blot analysis......Page 394
ELISA......Page 395
Link Between Advanced Glycation and ER Stress
......Page 396
Hypoxia......Page 397
ROS (oxidative stress)......Page 398
Aging......Page 399
Antioxidants......Page 400
Curcumin......Page 402
Antihypertensive drugs......Page 403
References......Page 404