Elsevier S & T

Small GTPases in Disease, Part A

Balch, W. E.

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Table of contents
  • Contentsv
  • Contributorsxv
  • Volumes in Seriesxxiii
  • Chapter 1: Rab1b Silencing Using Small Interfering RNA for Analysis of Disease-Specific Function1
  • 1. Introduction2
  • 2. Materials for siRNA Silencing of Rab1b4
  • 3. Procedure for Transfection of siRNA4
  • 4. Titration of Optimal siRNA Concentration5
  • 5. SDS-PAGE Analysis of Rab1 Knockdown6
  • 6. SDS-PAGE Analysis of CFTR6
  • 7. Discussion8
  • References9
  • Chapter 2: Rab8-Optineurin-Myosin VI: Analysis of Interactions and Functions in the Secretory Pathwa11
  • 1. Introduction12
  • 2. Identification of Myosin VI-Binding Partners13
  • 3. Verification of Yeast Two-Hybrid Screen Results13
  • 4. Mammalian Two-Hybrid Assay14
  • 5. Protocol for Mammalian Two-Hybrid Assay17
  • 6. SEAP Secretion Assay18
  • 7. Measurement of SEAP Secretion in Fibroblasts from Myosin VI Knockout Mouse19
  • 8. Measurement of SEAP Secretion in siRNA Knockdown Cells20
  • 9. Protocol for SEAP Assay21
  • Acknowledgments22
  • References22
  • Chapter 3: Characterization of Rab27a and JFC1 as Constituents of the Secretory Machinery of Prostat25
  • 1. Introduction26
  • 2. Methods27
  • 3. Concluding Remarks38
  • Acknowledgments39
  • References39
  • Chapter 4: In Vitro Assays to Characterize Inhibitors of the Activation of Small G Proteins by Their41
  • 1. GEFs and Diseases: Inhibiting the Exchange Reaction by Small Molecules42
  • 2. Initial Screen of Inhibitors of SMG Activation44
  • 3. Determination of Inhibition Profile by Using Michaelis-Menten Formalism49
  • 4. Complementary Assays to Refine.Noncompetitive and Uncompetitive Mechanisms50
  • 5. Conclusion53
  • Acknowledgments54
  • References54
  • Chapter 5: Analysis of Small GTPase Function in Trypanosomes57
  • 1. Overview57
  • 2. Culturing59
  • 3. Transfection and Cloning of T. brucei60
  • 4. Vector Systems for Overexpression and RNA Interference64
  • 5. Cloning and Manipulation of Trypanosome Small GTPase Sequences69
  • 6. Trafficking Assays to Monitor Exocytosis and Endocytosis71
  • Acknowledgments73
  • References73
  • Chapter 6: Use of Dynasore, the Small Molecule Inhibitor of Dynamin, in the Regulation of Endocytosi77
  • 1. Introduction78
  • 2. Dynamin78
  • 3. Dynamin and the Actin Cytoskeleton80
  • 4. The "Chemical Genetics" Discovery Approach81
  • 5. Why Do We Need Interfering Small Molecules?82
  • 6. Synthesis of Dynasore83
  • 7. Storage Conditions for Dynasore85
  • 8. Expression, Purification, and Storage of Dynamin85
  • 9. Buffers and Reagents87
  • Acknowledgment90
  • References91
  • Chapter 7: Identification and Verification of Sro7p as an Effector of the Sec4p Rab GTPase95
  • 1. Introduction96
  • 2. Experimental Approach to the Identification of Sec4p Effectors97
  • 3. Purification of GST-Sec4, GST-Ypt1, and GST, and Nucleotide Exchange98
  • 4. Preparation of the Yeast Extract for Sec4p Affinity Chromatography99
  • 5. Sec4p Affinity Chromatography100
  • 6. Confirmation of Sro7p as a Sec4p Effector102
  • 7. Co-Immunoprecipitation of Sro7p with Sec4p105
  • References107
  • Chapter 8: Characterization of Rab18, a Lipid Droplet-Associated Small GTPase109
  • 1. Introduction110
  • 2. Cell Systems111
  • 3. Generation of Rab18 Constructs and Point Mutations114
  • 4. Expression of Heterologous Proteins in 3T3-L1 Adipocytes116
  • 5. Biochemical Isolation of LDs118
  • 6. Fluorescence Microscopy119
  • 7. Electron Microscopy122
  • 8. Summary126
  • Acknowledgments127
  • References127
  • Chapter 9: Analysis on the Emerging Role of Rab3 GTPase-Activating Protein in Warburg Micro and Mart131
  • 1. Introduction132
  • 2. Materials133
  • 3. Methods134
  • 4. Comments138
  • References138
  • Chapter 10: Identification and Characterization of JRAB/MICAL-L2, a Junctional Rab13-Binding Protein141
  • 1. Introduction142
  • 2. Materials143
  • 3. Methods146
  • 4. Comments151
  • References151
  • Chapter 11: A SMAP Gene Family Encoding ARF GTPase-Activating Proteins and Its Implication in Membra155
  • 1. Introduction156
  • 2. Experimental Procedures and Results160
  • References169
  • Chapter 12: In Vitro and In Vivo Analysis of Neurotrophin-3 Activation of Arf6 and Rac-1171
  • 1. Introduction171
  • 2. Methods177
  • 3. Cell Culture and Transfection178
  • 4. Determination of Actin-Rich Protrusion Formation178
  • Acknowledgment181
  • References181
  • Chapter 13: Methods for Analysis of rab27a/munc13-4 in Secretory Lysosome Release in Hematopoietic C185
  • 1. Introduction186
  • 2. Affinity Purification of rab27a Effector Proteins187
  • 3. Direct Binding Assay with Recombinant Proteins191
  • 4. Generation of an Antibody Against Munc13-4193
  • 5. Degranulation Assay in RBL-2H3 Cells196
  • 6. Limited Proteolysis of 35S-Labeled munc13-4198
  • Acknowledgments199
  • References200
  • Chapter 14: Analysis and Expression of Rab38 in Oculocutaneous Lung Disease203
  • 1. Introduction204
  • 2. Morphological Examination of Mouse Lungs205
  • 3. Characterization of Recombinant Rab38cht-Protein209
  • 4. Discussion213
  • 5. Conclusion214
  • Acknowledgments214
  • References215
  • Chapter 15: Analysis of Rab1 Function in Cardiomyocyte Growth217
  • 1. Introduction218
  • 2. Generation of Adenoviruses Expressing Rab1 GTPase219
  • 3. Analysis of Adenovirus-Driven Rab1 Expression in Cardiomyocytes220
  • 4. Effect of Rab1 Infection on the Total Protein Synthesis in Neonatal Cardiomyocytes223
  • 5. Effect of Rab1 Infection on the Cell Size and Sarcomeric Organization of Cardiomyocytes223
  • 6. Cardiac Phenotypes of Transgenic Mice Overexpressing Rab1a in the Myocardium224
  • Acknowledgment225
  • References225
  • Chapter 16: Regulation of the Trafficking and Function of G Protein-Coupled Receptors by Rab1 GTPase227
  • 1. Introduction228
  • 2. Materials229
  • 3. Effects232
  • 4. Experimental Results and Concluding Remarks234
  • Acknowledgments236
  • References237
  • Chapter 17: Regulation of Endosome Dynamics by Rab5 and Huntingtin-HAP40 Effector Complex in Physiol239
  • 1. Introduction240
  • 2. Assays for Functional Characterization of Rab5, Htt, and HAP40243
  • 3. Spin-Down Binding Assays244
  • 4. In Vitro Motility assays251
  • Acknowledgments255
  • References255
  • Chapter 18: Biochemical and Biophysical Analyses of Ras Modification by Ubiquitin259
  • 1. Introduction260
  • 2. RAS Ubiquitination Monitored by Immunoprecipitation261
  • 3. RAS Ubiquitination Monitored by Ni-NTA Affinity Chromatograhy267
  • 4. RAS Ubiquitination Monitored by BRET in Living Cells268
  • 5. Concluding Remarks274
  • Acknowledgments275
  • References275
  • Chapter 19: Biochemical Characterization of Novel Germline BRAF and MEK Mutations in Cardio-Facio-Cu277
  • 1. Introduction278
  • 2. Methods279
  • 3. Results and Discussion282
  • Acknowledgments287
  • References287
  • Chapter 20: Biochemical and Biological Characterization of Tumor-Associated Mutations of p110alpha291
  • 1. Canonical Pathway292
  • 2. Cancer Link293
  • 3. Cancer-Specific Hot-Spot and Rare Mutations in p110alpha293
  • 4. Oncogenicity of Nonalpha Isoforms297
  • 5. Functional Importance of PIP3298
  • 6. Involvement of Ras in PI3K Signaling299
  • 7. Oncogenicity of Rheb300
  • 8. Conclusion301
  • Acknowledgments301
  • References302
  • Chapter 21: Characterization of the Rheb-mTOR Signaling Pathway in Mammalian Cells: Constitutive Act307
  • 1. Introduction308
  • 2. Methods310
  • 3. Conclusion317
  • References318
  • Chapter 22: Characterization of RalB-Sec5-TBK1 Function in Human Oncogenesis321
  • 1. Introduction322
  • 2. Methods323
  • 3. Conclusions328
  • Acknowledgments328
  • References329
  • Chapter 23: High-Throughput Lung Cancer Cell Line Screening for Genotype-Correlated Sensitivity to a331
  • 1. Introduction332
  • 2. Methods333
  • 3. Conclusions340
  • References340
  • Chapter 24: In Vitro Signaling by MAPK and NFkappaB Pathways Inhibited by Yersinia YopJ343
  • 1. Introduction343
  • 2. The in Vitro Signaling Assay345
  • 3. Materials and Methods346
  • 4. Conclusion352
  • Acknowledgments353
  • References353
  • Chapter 25: Biological and Biochemical Characterization of Anthrax Lethal Factor, a Proteolytic Inhi355
  • 1. Introduction356
  • 2. Materials and Methods357
  • 3. Concluding Remarks363
  • Acknowledgments363
  • References364
  • Chapter 26: Genetic Analyses of the Role of RCE1 in RAS Membrane Association and Transformation367
  • 1. Introduction368
  • 2. Establishing that Human Rce1 Is a CAAX Endoprotease370
  • 3. Rce1 Knockout Mice371
  • 4. Analyzing RAS Function in Rce1-/-MEFs371
  • 5. Absence of CAAX Endoprotease Activity in Rce1-Deficient Cells371
  • 6. Documenting an Accumulation of Uncleaved Rce1 Substrates in Rce1-Deficient Cells372
  • 7. Altered Electrophoretic Migration of RAS Proteins in Rce1-/-Fibroblasts374
  • 8. Methylation of RAS Proteins in Rce1-/-Fibroblasts374
  • 9. Subcellular Fractionation and Localization of RAS Proteins in Rce1-/-MEFs376
  • 10. Defining the Impact of Rce1 Deficiency on MAP Kinase Activation in Rce1-/-Fibroblasts377
  • 11. Impact of Endoproteolysis on the Binding of K-RAS to Microtubules378
  • 12. Defining the Impact of Rce1 Deficiency on Cell Growth and on Transformation by Activated Forms o378
  • 13. Effects of an Rce1 Excision on the Growth of RAS-Transfected Cells in Soft Agar and in Nude Mice379
  • 14. Analyzing the Functional Importance of Rce1 in the Liver and Heart381
  • 15. Minimal Effects of Rce1 Deficiency on the Hematopoietic System in Mice384
  • 16. Rce1 Deficiency Accelerates the Development of a K-RAS-Induced Myeloproliferative Disease385
  • References387
  • Chapter 27: Characterization of Kras-Mediated Pancreatic Tumorigenesis in Zebrafish391
  • 1. Introduction392
  • 2. Targeted Expression of eGFP and eGFP-KrasG12V in the Developing and Adult Zebrafish Exocrine Panc394
  • 3. Generation of Transgenic Zebrafish with Recombinant BAC DNA Constructs396
  • 4. Analysis of the Effects of eGFP-KrasG12V on Cellular Differentiation in the Zebrafish Embryonic P397
  • 5. Identification of Tumors Expressing eGFP-KrasG12V406
  • 6. Histologic Evaluation of Solid Tumors408
  • 7. Brief Remarks on the Diagnosis of Malignant Pancreatic Tumors in Zebrafish410
  • 8. Characterization of Protein Expression and RNA Expression in Zebrafish Tumors by Immunolabeling a411
  • References414
  • Chapter 28: Genetic Modeling of Ras-Induced Human Rhabdomyosarcoma419
  • 1. Introduction420
  • 2. Materials and Methods421
  • 3. Concluding Remarks425
  • References426
  • Author Index429
  • Subject Index455
Book details
  • Vendor Elsevier S & T
  • SKU 9780123739681
  • ISBN-13 9780080877600
  • Author Balch, W. E.
  • Category Science
  • Subject Molecular Biology

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The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 400 volumes (all of them still in print), the series contains much material still relevant today—truly an essential publication for researchers in all fields of life sciences.


Methods in Enzymology is now available online at ScienceDirect — full-text online of volumes 1 onwards. For more information about the Elsevier Book Series on ScienceDirect Program, please visit:
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This volume is the first of two planned volumes on the topic of small GTPases and their role in disease.