Regulators and effectors of small GTPases: Ras family

Introduction......Page 40
A Database of Ras Superfamily Proteins and Their Regulators......Page 41
References......Page 47
Real-Time In Vitro Measurement of Intrinsic and Ras GAP-Mediated GTP Hydrolysis......Page 49
Introduction......Page 50
PBP Preparation......Page 52
Labeling the PBP withMDCC......Page 55
Preparation of Small GTPases Complexed with GTP......Page 56
Measurement of GTPase Activity......Page 57
Data Analysis......Page 60
References......Page 61
Schwann Cell Preparation from Single Mouse Embryos: Analyses of Neurofibromin Function in Schwann Cells......Page 63
Introduction......Page 64
Solutions, Media,* and Supplies......Page 66
Schwann Cell Isolation from E12.5 Mouse Embryos......Page 67
Nf1-/-TXF Protocol......Page 69
Characterization of Phenotypes of Schwann Cells Derived from Wild-Type and Nf1 Mutant Mice......Page 70
References......Page 72
Introduction......Page 75
GTPase Activity Assays......Page 77
Detection of Protein-Bound Nucleotides by Thin-Layer Chromatography......Page 79
Characterization of a Bacterial Lysate Component with GTPase-Stimulating Activity......Page 80
Purification and Identification of the Protein Responsible for Lysate Activity......Page 81
GTPase-Inactivating Activity of NDK Is Specific to Mutant Ras Enzymes......Page 82
Nucleotides Do Not Detectably Dissociate from Ras During the Reaction with NDK......Page 83
NDK Catalyzes the Reverse Reaction: Conversion of Ras-GDP to Ras-GTP......Page 84
Conclusions......Page 86
References......Page 87
Introduction......Page 88
Materials......Page 89
Methods......Page 90
Data Analysis......Page 91
Method......Page 92
Data Analysis......Page 93
Principles......Page 94
Conclusion and Discussion......Page 95
References......Page 96
Overview......Page 97
Preparation of Chimeric GST-PAK1-RBD Protein for Cdc42 \"Pull-Down\"......Page 98
Retroviral Transduction of B Cell Lines......Page 99
AND-34-Induced Cdc42 and Rac Activation in MCF-7 Cells......Page 100
Akt In Vitro Kinase Assay......Page 101
Measurement of R-Ras GTP Levels......Page 102
References......Page 104
Introduction......Page 106
Protein Expression......Page 109
Binding To Phosphoinositides......Page 110
Ca2+-Mediated Association With Phospholipids......Page 111
Ras Pull-Down Assay......Page 112
Preparation of the Double-Stranded DNA Template......Page 114
Mutant Strand Synthesis Reaction......Page 115
Use of siRNA to Suppress Endogenous GAP1 Proteins......Page 116
Processing of Fixed Cells for Immunofluorescence and Epifluorescence......Page 117
Fixed and Live Cell Imaging......Page 118
Live Cell Imaging of the Ras GAP Activity of GAP1 Proteins......Page 120
References......Page 122
Introduction......Page 125
Ras Guanine Nucleotide Releasing Proteins (RasGRPs)......Page 126
Expression of RasGRP Proteins in Cells by Transient Transfection......Page 131
Expression of RasGRP Proteins in Cells by Retroviral Infection......Page 132
Measurement of Ras Activity in Response to RasGRP Expression and Activation by a Diacylglycerol Analog......Page 134
Subcellular Localization of RasGRP Proteins......Page 138
References......Page 139
Introduction......Page 142
Ras and Rap1 Activation of PLCepsiv Assessed by Transient Coexpression of Their Constitutive Active Mutants with PLCepsiv in COS-7 Cells......Page 144
Construction of BaF3 Transfectants Expressing the Mutant PDGF Receptor and PLCepsiv......Page 145
Assay for PLCepsiv Lipase Activity by Measuring Intracellular IP3 Levels by a Radioreceptor Assay Method......Page 146
Examples of Assay for Ras and Rap1 Activation of PLCepsiv Lipase Activity......Page 147
References......Page 149
Background......Page 151
Production and Purification of GST Fusion Proteins......Page 152
Ras Protein Binding Assay......Page 153
SH3 Binding Assays......Page 154
In Vivo Exchange Assay......Page 155
Binding of RalGPS PH Domain to Phospholipids......Page 156
References......Page 157
Introduction......Page 158
Expression Constructs......Page 160
Ral Dissociation Assay......Page 161
Signaling Assays......Page 163
In Vivo Expression of Rabbit Rgr......Page 165
Real-Time PCR Screening......Page 167
Purification of GST-Tagged HRgr protein......Page 168
Summary......Page 169
References......Page 170
Analysis of Ras Activation in Living Cells with GFP-RBD......Page 172
Introduction......Page 173
Cloning......Page 178
Cell Culture and Transfection......Page 179
Imaging and Stimulation......Page 181
Post-acquisition Image Analysis......Page 182
General Considerations......Page 183
Bystander FRET Assay......Page 184
Acknowledgments......Page 186
References......Page 187
Introduction......Page 188
Icmt Enzymatic Activity Assay......Page 190
Analyzing Expression of Icmt by RT-PCR......Page 191
Transfection of Cells with a GFP‐K‐Ras Fusion Construct\0......Page 192
Creation of Icmt-Deficient Mice and Cell Lines......Page 193
Is Icmt Responsible for the Carboxyl Methylation of the Ras Proteins?......Page 194
Is Icmt the Only Enzyme Capable of Methylating Isoprenylated Cysteine Residues?......Page 196
Is Icmt Responsible for Carboxyl Methylation of the CXC Class of Rab Proteins?......Page 197
Impact of Icmt on Ras Transformation......Page 198
Pharmacologic Inhibition of Icmt......Page 199
Acknowledgments......Page 200
References......Page 201
Characterization of the Activation of the Rap-Specific Exchange Factor Epac by Cyclic Nucleotides......Page 204
Introduction......Page 205
General Overview......Page 206
Purification of Epac......Page 209
Purification of Rap and mGDP-Loading......Page 211
Appendix......Page 212
Acknowledgments......Page 217
References......Page 218
Introduction......Page 219
Isolation from Escherichia coli......Page 221
Isolation of Bacmid DNA......Page 222
Generation of Baculovirus......Page 223
Protein Isolation from Baculovirus-Infected Cells......Page 224
Measuring GEF Activity......Page 225
Preparation of Cell Extracts for Identification of PDZ-GEF Activating Compounds......Page 226
Isolation of PDZ-GEF-RCBD Protein......Page 227
Generation of Stable Cell Lines......Page 228
Discussion......Page 229
References......Page 230
Introduction......Page 232
In Vitro Interactions with Purified Proteins......Page 233
Purification of GST Effectors......Page 234
Purification of His-RFGs and Standardization of Stocks to the Same Amounts of \"Active\" GTPase......Page 235
Binding Reaction......Page 236
Interactions in Mammalian Cells......Page 237
References......Page 239
Introduction......Page 240
H-Ras Effector Domain Mutants......Page 241
Constitutively Activated Mutant Effectors......Page 242
Constitutively Activated Effector Substrates......Page 246
Reagents for Assessment of Effector Necessity......Page 247
Pharmacological Inhibitors......Page 248
Interfering RNA (RNAi)......Page 250
Generation of Infectious Retrovirus......Page 252
Verification of Effector Expression and Activation......Page 254
Analysis of Effector Function in Ras-Mediated Morphological and Growth Transformation of ROSE Ovarian Epithelial Cell......Page 256
References......Page 258
Introduction......Page 263
PCR Primers......Page 265
PCR......Page 266
DNA Sequencing......Page 267
Laboratory Information System......Page 268
References......Page 269
KSR Regulation of the Raf-MEK-ERK Cascade......Page 270
Introduction......Page 271
Analyzing the ERK Scaffolding Activity of KSR Using the Xenopus laevis Oocyte Meiotic Maturation Assay......Page 272
Buffers......Page 273
Isolation of Stage VI Oocytes......Page 275
Injection and Monitoring of Oocytes......Page 276
Determination of Exogenous KSR and RasV12 Protein Expression......Page 277
Determining the Activation State of MEK and ERK......Page 278
Analyzing the ERK Scaffolding Activity of KSR in Mammalian Cells......Page 279
Immunofluorescent Staining......Page 280
References......Page 281
Introduction......Page 284
Cell Culture......Page 286
Antibodies......Page 287
RNA Interference of Endogenous IMP Transcripts......Page 288
Antibodies......Page 289
Antibodies......Page 290
Antibodies......Page 291
KSR1 Hyperphosphorylation and Solubility......Page 292
References......Page 293
RKIP Functions......Page 295
RKIP Gene Family......Page 297
Coimmunoprecipitation of Raf-1 and RKIP......Page 298
Measuring the Influence of RKIP on Raf Kinase Activity......Page 300
Production of Recombinant GST-tagged Raf Proteins in Sf-9 Insect Cells......Page 301
Immunohistochemistry Protocol......Page 303
Commercially Available RKIP Antibodies......Page 304
References......Page 305
Introduction......Page 307
Transfection......Page 309
Trypsin‐Primed Transfection\0......Page 310
Control siRNAs......Page 312
Limitations......Page 313
Application to Targets Supporting Oncogenic Transformation......Page 314
References......Page 315
Introduction......Page 317
Ras-Induced Skin Carcinogenesis......Page 319
Protocol for Two‐stage DMBA/TPA Carcinogenesis\0......Page 321
Proliferation and Apoptosis Assays In Vivo......Page 322
In Vitro Analysis of Tiam1/Rac Signaling......Page 323
Isolation of Skin Tumor Cells......Page 325
Isolation of Keratinocytes......Page 326
References......Page 327
Phospholipase Cepsi Guanine Nucleotide Exchange Factor Activity and Activation of Rap1......Page 330
Introduction......Page 331
Materials and Methods......Page 332
Materials and Methods......Page 333
Results......Page 335
Materials and Methods......Page 336
Results......Page 337
References......Page 338
Nore1 and RASSF1 Regulation of Cell Proliferation and of the MST1/2 Kinases......Page 340
Introduction......Page 341
Nore1/RASSF1 Polypeptides Inhibit Cell Proliferation and Are Candidate Tumor Suppressors......Page 343
RASSF1A/Nore1A Inhibit G1 Progression Independently of Ras-Like GTPases......Page 345
RASSF1 and Nore1A Participate in Ras-Induced Apoptosis......Page 346
RASSF1A Associates with the Microtubular Apparatus and Controls APC/Cyclosome Activation in Early Mitosis......Page 347
The Nore1/RASSF Polypeptides All Bind the Class II GC Kinases, MST1 and MST2......Page 348
The MST1/2 Kinases Mediate Ras-Induced Apoptosis......Page 351
A Phosphopeptide-Specific Antibody Specific for Activated MST1/2......Page 352
Quantitative Assay of MST1/2 Activation In Vitro......Page 353
Estimation of MST Activation In Vivo......Page 354
Effect of NORE1/RASSF1 on MST Activation In Vitro and In Vivo......Page 355
References......Page 356
Introduction......Page 361
Ras Binding in Cells......Page 362
Ras Binding In Vitro......Page 363
Fluorescent Apoptosis Assays......Page 364
Cell Cycle Assays......Page 365
Tubulin Stabilization Assays......Page 366
Motility Assays......Page 367
Genetic Instability......Page 368
Inhibition of RASSF1A Function by ShRNA/SiRNA......Page 369
References......Page 371
RAS and the RAIN/RasIP1 Effector......Page 373
Expression Constructs......Page 374
Cell Lines......Page 376
Cell Culture and DNA Transfer......Page 377
Procedure......Page 378
Visualization of the RAIN-Ras Interaction In Vivo......Page 379
Cell Culture and DNA Transfer......Page 380
Endogenous RAIN Expression in Endothelial Cells......Page 381
Reagents for Immunostaining......Page 383
Immunostaining Procedure......Page 384
References......Page 385
Introduction......Page 387
Measurement of GTP/GDP Ratios for RAB5 Proteins......Page 389
Measurement of Receptor Endocytosis Levels......Page 390
ABL-Mediated Changes in Cell Function......Page 391
RIN Family Evolution......Page 392
RIN Genes Have Distinct Patterns of Expression......Page 393
References......Page 395
Introduction......Page 397
Yeast Two-Hybrid System or Interaction Trap......Page 398
Yeast Two-Hybrid Interaction Trap Assay......Page 399
In Vitro Protein-Protein Interaction by Pull-Down Assays......Page 400
Expression and Purification of GST-Rap1 Active and Inactive Mutant Fusions from E. coli......Page 401
Generation of S35 [Methionine]-Labeled RIAM Protein......Page 402
Transient Transfection of RIAM in T Cells by Electroporation......Page 403
Adhesion Assay to Immobilized Substrate......Page 404
Inhibition of Cell Adhesion to ICAM-1 and Fibronectin......Page 405
Intracellular Localization of Active Rap1......Page 407
Intracellular Staining......Page 408
References......Page 409
Introduction......Page 411
Rap1 Localization in Epithelial Cells......Page 412
Time Lapse......Page 413
Production of Fc‐Tagged Extracellular Domain of E‐Cadherin (hE/Fc)\0......Page 414
Coupling of hE/Fc to Polystyrene Beads......Page 416
Imaging GFP‐Rap1 Localization and hE/Fc‐Coated Beads\0......Page 417
Rap Activation by Cadherin-Mediated Adhesion......Page 418
RalGDS Beads......Page 419
Pull‐Down\0......Page 420
Determination of Rap Role in Cadherin-Dependent Cell-Cell Contacts......Page 421
Microinjection......Page 422
References......Page 423
Introduction......Page 425
ROSE 199 and KRAS/Transformed A2/5 Cells as a Model for the Tumorigenic Conversion of Ovarian Surface Epithelium......Page 426
Total RNA Isolation, mRNA Preparation, and cDNA Synthesis......Page 427
Suppression Subtractive Hybridization and Establishment of Subtracted cDNA Libraries......Page 428
Sequence Analysis of Subtracted Libraries and Validation of Differential Expression......Page 430
Preparation of Slides and Spotting......Page 431
Labeling Procedure......Page 433
cDNA Hybridization and Washing......Page 434
3DNA Reagent Hybridization and Washing......Page 435
Discussion......Page 436
Acknowledgments......Page 438
References......Page 439
Introduction......Page 440
Background and General Considerations......Page 441
Fragment Synthesis and Construct Preparation......Page 442
Image Acquisition and Analysis......Page 443
Mapping Signaling Complexes, Pathways, and Networks......Page 444
Probing Pathway Architecture and Signaling Dynamics......Page 446
HTS and Probing Novel Targets......Page 450
Conclusions......Page 451
References......Page 452
Ras Up-Regulation of Cyclooxygenase-2......Page 454
Introduction......Page 455
Examination of Transcriptional, Posttranscriptional, and Translational Regulation of COX-2 Expression......Page 456
Comments......Page 457
Stable Transfection of RIE-iRas Cells and Assaying for COX-2 Reporter Activity......Page 458
Ras Activation Assays and Western Blotting......Page 459
Comments......Page 460
References......Page 462
Introduction......Page 464
The Model System......Page 465
TM mRNAs in Normal and ras-Transformed Fibroblasts......Page 466
Downregulation of TM Proteins by Ras......Page 467
Methods of Sample Preparation......Page 468
Analysis of TMs by Immunoblotting-Antibody Specificity......Page 469
Downregulation of TMs and Cytoskeletal Disruption......Page 471
Role of TMs in Regulation of Cytoskeleton and Cell Phenotype......Page 472
References......Page 473
Introduction......Page 477
Transient and Stable Transfection......Page 479
Western Blot Analysis for Protein Expression......Page 480
Transferring Proteins onto a Membrane......Page 482
Northern Blot Analysis......Page 484
Preparation of RNA Gel......Page 485
Assay for Promoter Down-Regulation by Measurement of Reporter Activity......Page 487
Flow Cytometry......Page 488
Cell Cycle Synchronization......Page 489
Analysis of Transformation by Ras......Page 490
3[H] Thymidine Incorporation Assay for Cell Proliferation......Page 491
Focus Formation Assay......Page 492
Analysis of Apoptosis......Page 493
Annexin V-Based Analysis of Apoptosis......Page 494
Analysis of Apoptosis with DAPI......Page 495
References......Page 496
Introduction......Page 498
Drosophila......Page 499
Fission Yeast......Page 505
References......Page 507
Introduction......Page 510
Imprinted Genes Can Contribute to Human Carcinogenesis......Page 511
ARHI Encodes a 26-kDa GTPase with Homology to Ras......Page 512
ARHI Is Dramatically Downregulated in Most Ovarian and Breast Cancers......Page 515
Expression from the Paternal Allele of ARHI Can Be Lost Through LOH, CpG Methylation, and Transcriptional Regulation......Page 516
Overexpression of ARHI in Cancer Cells Induces Caspase-Independent, Calpain-Dependent Apoptosis, Whereas Physiological Expression of ARHI Induces Autophagy......Page 518
Conclusion......Page 520
References......Page 521
Gem Protein Signaling and Regulation......Page 524
Introduction......Page 525
Inhibition of Rho Kinase-Mediated Cytoskeletal Reorganization......Page 526
Effect on Stress Fiber and Focal Adhesion Formation......Page 527
Immunofluorescent Staining and Confocal Microscopy......Page 528
Transient Transfection......Page 529
Redirection of Rho Kinase Substrate Specificity......Page 530
TCA Precipitation and Urea Extraction......Page 531
Procedure......Page 532
LIM-Kinase 1 Activity Assay......Page 533
In Vitro Kinase Assay......Page 534
Inhibition of Voltage-Gated Calcium Channel Activity......Page 535
Hormone Secretion Assay......Page 536
Regulation of Gem Function by Posttranslational Modification......Page 537
References......Page 538
Introduction......Page 540
Mammalian Expression Vectors for Wild-Type and Mutant Rem Proteins......Page 542
Generation of Recombinant GST-Rem......Page 543
\"Far Western\" Interaction Cloning to Identify Rem Binding Proteins......Page 544
In Vitro Binding Reactions......Page 545
HEK293 Cell Culture......Page 546
Reagents For Transient Transfection......Page 547
Analysis of L-Type Calcium Channel Function......Page 548
Electrophysiological Recordings in HEK293 Cells......Page 549
Electrophysiology......Page 550
Solutions for Electrophysiology......Page 552
References......Page 553
Introduction......Page 555
Mammalian Expression Vectors for Wild-Type and Mutant Rit Proteins......Page 556
PC6 Cell Culture......Page 557
Transfection Procedure......Page 559
Signaling Analyses......Page 560
Raf Kinase Assays......Page 561
p38 Mitogen-Activated Protein Kinase Assay......Page 563
Analysis of Rit-B-Raf Interaction by In Vitro Binding Assay......Page 564
Purification of Glutathione S-Transferase RGL3-RBD Protein......Page 565
Detection of Rit-GTP Levels Using GST-RGL3-RBD as an Activation-Specific Probe......Page 566
Yeast Two-Hybrid Analysis of Rit Effector Interactions......Page 567
References......Page 568
Identification and Gene Structure......Page 569
Relationship to Ras Proteins......Page 571
Molecular Constructs and Mutants of RERG......Page 572
Tumors and Tumor-Derived Cell Lines......Page 573
Analysis of RERG Protein Subcellular Localization......Page 576
Biochemical Properties and Function......Page 577
RERG as Suppressor of Growth and Tumor Formation......Page 579
Summary......Page 581
References......Page 582
Introduction......Page 584
kappaB-Ras Expression and Purification......Page 587
NF-kappaB DNA Binding Inhibition Assay......Page 589
Competition Assay......Page 590
References......Page 591
Introduction: The Rhes/Dexras Subfamily......Page 592
Analysis of Rhes Nucleotide Loading......Page 593
Analysis of Rhes Nucleotide Loading by Effector Pull-Down......Page 595
Effect of Rhes on the cAMP Pathway......Page 597
References......Page 599
Rheb Activation of mTOR and S6K1 Signaling......Page 600
Introduction......Page 601
Analysis of Protein Phosphorylation......Page 602
Utilization of Inhibitory Drugs......Page 604
GTPase-Activating Protein (GAP) Assay......Page 605
Purification and Preparation of Flag-TSC1/2......Page 606
GAP Assay......Page 607
Cell Transfection......Page 608
Conclusion......Page 609
References......Page 610
Introduction......Page 614
Phosphorylation of Oligonucleotides......Page 616
Annealing Oligonucleotides......Page 617
Generation of pSUPER.retro.siRNA Virus for Stable Expression of siRNA to Define the Role of Mutant Ras......Page 618
Production of Infectious Virus in 293T Cells: Host Cell Preparation......Page 619
Plasmid DNA Preparation and 293T Host Cell Transfection......Page 620
Target Cell Preparation and Infection......Page 621
Consequences of Suppression of K-Ras (12V) Expression......Page 622
Generation of Modified Vectors for Inducible Expression of shRNAs......Page 625
Establishment and Analysis of Human Tumor Cell Lines for Inducible shRNA-Mediated Silencing of Mutant K-Ras(12V) Expression......Page 626
Analyses of Ras Effector Function and Oncogenesis......Page 629
References......Page 631
Introduction......Page 633
General Considerations......Page 635
Transfecting Cells for FTI and GGTI Treatment......Page 636
Analysis of FTI and GGTI Effects on Ras and Rho GTPase Localization......Page 637
Using Prenylation Inhibitors to Abrogate Focus-Forming Activity......Page 638
Using Prenyltransferase Inhibitors to Disrupt Colony-Forming Activity in Soft Agar......Page 641
Preparation of FTS Stock and Working Solutions......Page 644
FTS Treatment of Ras‐transformed Cells for Growth Inhibition and Other Endpoints\0......Page 645
Evaluation of Ras Protein Levels on FTS Treatment......Page 647
Generation of Cell Lysates Coexpressing Rhogdi and Rho Proteins......Page 648
Performing the Immunoprecipitation......Page 649
Detecting the Coimmunoprecipitated Proteins......Page 650
Visualization of Rhogdi Effects on Rho Localization......Page 651
Concluding Remarks......Page 653
References......Page 654
Sorafenib (BAY 43-9006, Nexavarreg), a Dual-Action Inhibitor That Targets RAF/MEK/ERK Pathway in Tumor Cells and Tyrosine Kinases VEGFR/PDGFR in Tumor Vasculature......Page 656
Introduction......Page 657
Phospho-ERK Bio-Plex Immunoassay......Page 659
Immunohistochemistry......Page 661
Sorafenib Inhibits MEK and ERK Phosphorylation in Cancer Cells, Independent of K-Ras and B-RAF Mutational Status......Page 662
Inhibition of ERK Phosphorylation by Sorafenib in MDA-MB-231 Tumors......Page 666
References......Page 668
Introduction......Page 672
Developing Yeast Two-Hybrid Strains Permeable for Small Molecular Weight Compounds......Page 675
Permeability Agar-Plate Screening Protocol......Page 677
Developing HTS for Small Molecular Weight Inhibitors of Ras-Raf Interaction in Highly Permeable Yeast Two-Hybrid System......Page 678
Analysis of Selectivity of MCP Compounds by Liquid beta-Galactosidase Yeast Two-Hybrid Assay......Page 680
Liquid beta-Gal Assay Protocol......Page 681
Developing a Robust Decision Tree for Compound Selection and Optimization: Summary of Assays Applied......Page 682
Ras-Raf Interaction Inhibitors Downregulate Signaling Through the MAPK Pathway......Page 684
Refining the Mechanistic Understanding of MCP Activity and Optimization of MCPs for the Clinic......Page 685
Discussion......Page 686
References......Page 687
Introduction......Page 690
Chemical Synthesis......Page 692
Gel Mobility Shift Assay for Ras and Hdj-2 Farnesylated Proteins......Page 693
Global Detection and Affinity Purification of Azido-Farnesylated Proteins......Page 695
References......Page 697
A Genetically Defined Normal Human Somatic Cell System to Study Ras Oncogenesis In Vivo and In Vitro......Page 699
Introduction......Page 700
Methods......Page 702
Materials......Page 703
Materials......Page 705
Concluding Remarks......Page 706
References......Page 708
Introduction......Page 710
Overview of Biological Results Obtained with the In Vitro Thyroid Model and Effect of Mutant RAS on Thyroid-Specific Differentiation......Page 711
RAF-MAPK Pathway......Page 713
PI3K Pathway......Page 714
Differential Response of Human Fibroblasts and Thyrocytes to Mutant Ras Oncoprotein......Page 715
Methods 1: Disaggregation of Thyroid Tissue to Produce Primary Monolayer Cultures of Follicular Epithelium......Page 717
Production of the Amphotropic Vector......Page 718
Methods for Transient Expression of Mutant Ras in Thyroid Epithelial Cells......Page 719
References......Page 720
Introduction......Page 723
Isolation and Culture of Normal Human Ovarian Surface Epithelia Cells......Page 724
Cell Passage......Page 726
Growth Media......Page 727
Distinguishing Human Ovarian Surface Epithelial Cells from Fibroblasts......Page 728
Materials and Reagents......Page 730
Criteria for Immortalization......Page 731
Packaging Cell Lines......Page 732
Materials and Reagents......Page 733
Tumorigenicity in Mice......Page 734
Histopathological Analysis of Tumors......Page 735
References......Page 737
Introduction......Page 740
Modeling Oncogenic K-ras in Mice......Page 741
Genomic DNA Preparation......Page 743
PCR Genotyping......Page 744
RAS-GTP, Total RAS, and K-rasG12D from Murine Pancreas......Page 745
Preparation of MEFs......Page 746
Infection of MEFs with Recombinant Adenoviruses That Express Cre Recombinase......Page 748
Molecular Evidence of LSL-K-ras* Recombination and Expression......Page 749
Senescence-Associated Beta-Galactosidase Activity in Oncogenic ras-Expressing MEFs......Page 750
Colony Formation and Focus Formation in K-rasG12D MEFs......Page 751
Protocol......Page 752
References......Page 753
Introduction......Page 755
Generating ER-Ras Fusions......Page 757
Conditional Ras Activation in Primary Human Keratinocytes in Culture......Page 758
Modulating Ras Activity In Vivo......Page 761
Concluding Remarks......Page 764
References......Page 765
Introduction......Page 767
Primary Pancreatic Duct Epithelial Cell (PDEC) Isolation......Page 768
Two-Dimensional Cultures......Page 770
Three-Dimensional Cultures on Reconstituted Basement Membrane......Page 771
Overlay Method......Page 772
Immunofluorescence Staining of Matrigel-Overlaid PDEC......Page 773
References......Page 774
Introduction......Page 775
UAS Lines......Page 776
Sev-Gal4......Page 777
FLP/FRT Recombination to Generate Cells Mutant for Ras Pathway Components......Page 778
Reagents for Assessing Differentiation, Proliferation, and Death......Page 781
TUNEL (Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-end Labeling)......Page 782
References......Page 783