Includes experimental approaches to the complexity of the biological functions of RasGTPases
Provides step-by-step detail essential for reproducible results
Contains key notes and implementation advice from the experts
Featuring experimental approaches that shed light on the complexity of Ras GTPase biological functions, Ras Signaling: Methods and Protocols contains general overviews and detailed applications of both well-established and recently developed research techniques, including biochemical, biophysical, molecular biology, genetic and behavioral approaches, advanced high resolution fluorescence and electron microscopy imaging and "omics” technologies. Through this, the detailed volume provides information on expression, post-translational modifications, subcellular localization and dynamics, regulatory mechanisms of upstream and downstream signaling pathways and ultimately, biological activities and functions of Ras GTPases in different model systems, including high and low eukaryotic organisms. Written in the highly successful Methods in Molecular Biology series format, chapters include brief introductions, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips on troubleshooting and avoiding known pitfalls.
Wide-ranging and authoritative, Ras Signaling: Methods and Protocols serves as an aid for investigators of different backgrounds and interests related to the multiple physiological and pathological functions of the large superfamily of Ras GTPases.
1. The Ras Superfamily of Small GTPases: The Unlocked Secrets
Luca Goitre, Eliana Trapani, Lorenza Trabalzini, and Saverio Francesco Retta
2. A Novel Method for the Production of Fully-Modified K-Ras 4B
Tanmay S. Chavan, James O. Meyer, Luke Chisholm, Malgorzata Dobosz, and Vadim Gaponenko
3. Metabolic Labeling of Ras with Tritiated Palmitate to Monitor Palmitoylation and Depalmitoylation
Frederick D. Tsai, Joseph P. Wynne, Ian M. Ahearn, and Mark R. Philips
4. Ubiquitination of Rac1 by Inhibitors of Apoptosis (IAPs)
Tripat Kaur Oberoi-Khanuja and Krishnaraj Rajalingam
5. Ras GTPases are Both Regulators and Effectors of Redox Agents
Elisa Ferro, Luca Goitre, Eva Baldini, Saverio Francesco Retta, and Lorenza Trabalzini
6. Biophysical and Proteomic Characterization Strategies for Cysteine Modifications in Ras GTPases
G. Aaron Hobbs, Harsha P. Gunawardena, and Sharon L. Campbell
7. Use of the Yeast Two-Hybrid Technology to Isolate Molecular Interactions of Ras GTPases
Elisa Ferro, Eva Baldini, and Lorenza Trabalzini
8. Screening for MAPK Modulators Using an In-Cell Western Assay
Simon Schnaiter, Beatrix Fürst, Johannes Neu, Frigyes Wáczek, László Orfi, György Kéri, Lukas A. Huber, and Winfried Wunderlich
9. Behavioral Methods for the Study of the Ras-ERK Pathway in Memory Formation and Consolidation: Passive Avoidance and Novel Object Recognition Tests
Raffaele d´Isa, Riccardo Brambilla, and Stefania Fasano
10. Functional Phosphoproteomics of Oncogenic KRAS Signaling
Putty-Reddy Sudhir and Jeou-Yuan Chen
11. Pull-Down Assay for Analysis of Integrin-Mediated Activation of Rap Proteins in Adherent Platelets
Gianni Francesco Guidetti and Mauro Torti
12. Combined Pulldown and Time-Lapse Microscopy Studies for Determining the Role of Rap1 in the Crosstalk between Integrins and Cadherins
Luca Goitre, Alessandro Morina, and Saverio Francesco Retta
13. Fluorescence Microscopy Study of Rap1 Subcellular Localization
Luca Goitre, Valentina Cutano, and Saverio Francesco Retta
14. An In Vitro System to Evaluate the Scaffold Function of the RalA Effector Protein RalBP1
David F. Kashatus
15. Analysis of the Rit Subfamily GTPase-Mediated Signaling and Neuronal Differentiation and Survival
Geng-Xian Shi and Catherine N. Kaminski
16. Immunofluorescence Methods in Studies of the GTPase RAN and Its Effectors in Interphase and in Mitotic Cells
Giulia Guarguaglini, Valeria de Turris, and Patrizia Lavia
17. High Resolution Scanning Electron Microscopy for the Imaging of Nuclear Pore Complexes and Ran-Mediated Transport
Lihi Shaulov, Boris Fichtman, and Amnon Harel
18. Effector Recruitment Method to Study Spatially Regulated Activation of Ras and Rho GTPases
Lauren P. Huff, Molly J. DeCristo, and Adrienne D. Cox
19. Real-Time Visualization and Quantification of Native Ras Activation in Single Living Cells
Christoph Biskup and Ignacio Rubio
20. Nanoclustering and Heterogeneous Membrane Diffusion of Ras Studied by FRAP and RICS Analysis
Camilo Guzmán, Maja Šolman, and Daniel Abankwa
21. Analyzing the Roles of Rho GTPases in Cancer Cell Migration with a Live Cell Imaging 3D-Morphology-Based Assay
Audrey Colomba and Anne J. Ridley
22. Analysis of Rho-GTPase-Induced Localization of Nanoscale Adhesions Using Fluorescence Nanoscopy
Annica K.B. Gad, Daniel Rönnlund, Jerker Widengren, and Pontus Aspenström
23. Yeast as a Model for Ras Signaling
Renata Tisi, Fiorella Belotti, and Enzo Martegani
24. Methods to Study the Ras2 Protein Activation State and the Subcellular Localization of Ras-GTP in Saccharomyces cerevisiae
Sonia Colombo and Enzo Martegani
25. RAS Proteins Signaling in the Early Metazoan Dictyostelium discoideum
Enrico Bracco and Barbara Pergolizzi
Featuring experimental approaches that shed light on the complexity of Ras GTPase biological functions, Ras Signaling: Methods and Protocols contains general overviews and detailed applications of both well-established and recently developed research techniques, including biochemical, biophysical, molecular biology, genetic and behavioral approaches, advanced high resolution fluorescence and electron microscopy imaging, and "omics” technologies. Through this, the detailed volume provides information on expression, post-translational modifications, subcellular localization and dynamics, regulatory mechanisms of upstream and downstream signaling pathways, and, ultimately, biological activities and functions of RasGTPases in different model systems, including high and low eukaryotic organisms. Written in the highly successful Methods in Molecular Biology series format, chapters include brief introductions, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Wide-ranging and authoritative,
Ras Signaling: Methods and Protocols serves as an aid for investigators of different backgrounds and interests related to the multiple physiological and pathological functions of the large superfamily of RasGTPases.
Featuring experimental approaches that shed light on the complexity of Ras GTPase biological functions, Ras Signaling: Methods and Protocols contains general overviews and detailed applications of both well-established and recently developed research techniques, including biochemical, biophysical, molecular biology, genetic and behavioral approaches, advanced high resolution fluorescence and electron microscopy imaging and "omics" technologies. Through this, the detailed volume provides information on expression, post-translational modifications, subcellular localization and dynamics, regulatory mechanisms of upstream and downstream signaling pathways and ultimately, biological activities and functions of Ras GTPases in different model systems, including high and low eukaryotic organisms. Written in the highly successful Methods in Molecular Biology series format, chapters include brief introductions, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips on troubleshooting and avoiding known pitfalls.
Wide-ranging and authoritative, Ras Signaling: Methods and Protocols serves as an aid for investigators of different backgrounds and interests related to the multiple physiological and pathological functions of the large superfamily of Ras GTPases.
The Ras Superfamily of Small GTPases: The Unlocked Secrets.- A Novel Method for the Production of Fully-Modified K-Ras 4B.- Metabolic Labeling of Ras with Tritiated Palmitate to Monitor Palmitoylation and Depalmitoylation.- Ubiquitination of Rac1 by Inhibitors of Apoptosis (IAPs).- Ras GTPases are Both Regulators and Effectors of Redox Agents.- Biophysical and Proteomic Characterization Strategies for Cysteine Modifications in Ras GTPases.- Use of the Yeast Two-Hybrid Technology to Isolate Molecular Interactions of Ras GTPases.- Screening for MAPK Modulators Using an In-Cell Western Assay.- Behavioral Methods for the Study of the Ras-ERK Pathway in Memory Formation and Consolidation: Passive Avoidance and Novel Object Recognition Tests.- Functional Phosphoproteomics of Oncogenic KRAS Signaling.- Pull-Down Assay for Analysis of Integrin-Mediated Activation of Rap Proteins in Adherent Platelets.- Combined Pulldown and Time-Lapse Microscopy Studies for Determining the Role of Rap1 in the Crosstalk Between Integrins and Cadherins.- Fluorescence Microscopy Study of Rap1 Subcellular Localization.- An In Vitro System to Evaluate the Scaffold Function of the RalA Effector Protein RalBP1.- Analysis of the Rit Subfamily GTPase-Mediated Signaling and Neuronal Differentiation and Survival.- Immunofluorescence Methods in Studies of the GTPase RAN and Its Effectors in Interphase and in Mitotic Cells.- High Resolution Scanning Electron Microscopy for the Imaging of Nuclear Pore Complexes and Ran-Mediated Transport.- Effector Recruitment Method to Study Spatially Regulated Activation of Ras and Rho GTPases.- Real-Time Visualization and Quantification of Native Ras Activation in Single Living Cells.- Nanoclustering and Heterogeneous Membrane Diffusion of Ras Studied by FRAP and RICS Analysis.- Analyzing the Roles of Rho GTPases in Cancer Cell Migration with a Live Cell Imaging 3D-Morphology-Based Assay.- Analysis of Rho-GTPase-Induced Localization of Nanoscale AdhesionsUsing Fluorescence Nanoscopy.- Yeast as a Model for Ras Signaling.- Methods to Study the Ras2 Protein Activation State and the Subcellular Localization of Ras-GTP in Saccharomyces cerevisiae.- RAS Proteins Signaling in the Early Metazoan Dictyostelium discoideum.
Inhaltsverzeichnis
1. The Ras Superfamily of Small GTPases: The Unlocked Secrets
Luca Goitre, Eliana Trapani, Lorenza Trabalzini, and Saverio Francesco Retta
2. A Novel Method for the Production of Fully-Modified K-Ras 4B
Tanmay S. Chavan, James O. Meyer, Luke Chisholm, Malgorzata Dobosz, and Vadim Gaponenko
3. Metabolic Labeling of Ras with Tritiated Palmitate to Monitor Palmitoylation and Depalmitoylation
Frederick D. Tsai, Joseph P. Wynne, Ian M. Ahearn, and Mark R. Philips
4. Ubiquitination of Rac1 by Inhibitors of Apoptosis (IAPs)
Tripat Kaur Oberoi-Khanuja and Krishnaraj Rajalingam
5. Ras GTPases are Both Regulators and Effectors of Redox Agents
Elisa Ferro, Luca Goitre, Eva Baldini, Saverio Francesco Retta, and Lorenza Trabalzini
6. Biophysical and Proteomic Characterization Strategies for Cysteine Modifications in Ras GTPases
G. Aaron Hobbs, Harsha P. Gunawardena, and Sharon L. Campbell
7. Use of the Yeast Two-Hybrid Technology to Isolate Molecular Interactions of Ras GTPases
Elisa Ferro, Eva Baldini, and Lorenza Trabalzini
8. Screening for MAPK Modulators Using an In-Cell Western Assay
Simon Schnaiter, Beatrix Fürst, Johannes Neu, Frigyes Wáczek, László Orfi, György Kéri, Lukas A. Huber, and Winfried Wunderlich
9. Behavioral Methods for the Study of the Ras-ERK Pathway in Memory Formation and Consolidation: Passive Avoidance and Novel Object Recognition Tests
Raffaele d'Isa, Riccardo Brambilla, and Stefania Fasano
10. Functional Phosphoproteomics of Oncogenic KRAS Signaling
Putty-Reddy Sudhir and Jeou-Yuan Chen
11. Pull-Down Assay for Analysis of Integrin-Mediated Activation of Rap Proteins in Adherent Platelets
Gianni Francesco Guidetti and Mauro Torti
12. Combined Pulldown and Time-Lapse Microscopy Studies for Determining the Role of Rap1 in the Crosstalk between Integrins and Cadherins
Luca Goitre, Alessandro Morina, and Saverio Francesco Retta
13. Fluorescence Microscopy Study of Rap1 Subcellular Localization
Luca Goitre, Valentina Cutano, and Saverio Francesco Retta
14. An In Vitro System to Evaluate the Scaffold Function of the RalA Effector Protein RalBP1
David F. Kashatus
15. Analysis of the Rit Subfamily GTPase-Mediated Signaling and Neuronal Differentiation and Survival
Geng-Xian Shi and Catherine N. Kaminski
16. Immunofluorescence Methods in Studies of the GTPase RAN and Its Effectors in Interphase and in Mitotic Cells
Giulia Guarguaglini, Valeria de Turris, and Patrizia Lavia
17. High Resolution Scanning Electron Microscopy for the Imaging of Nuclear Pore Complexes and Ran-Mediated Transport
Lihi Shaulov, Boris Fichtman, and Amnon Harel
18. Effector Recruitment Method to Study Spatially Regulated Activation of Ras and Rho GTPases
Lauren P. Huff, Molly J. DeCristo, and Adrienne D. Cox
19. Real-Time Visualization and Quantification of Native Ras Activation in Single Living Cells
Christoph Biskup and Ignacio Rubio
20. Nanoclustering and Heterogeneous Membrane Diffusion of Ras Studied by FRAP and RICS Analysis
Camilo Guzmán, Maja solman, and Daniel Abankwa
21. Analyzing the Roles of Rho GTPases in Cancer Cell Migration with a Live Cell Imaging 3D-Morphology-Based Assay
Audrey Colomba and Anne J. Ridley
22. Analysis of Rho-GTPase-Induced Localization of Nanoscale Adhesions Using Fluorescence Nanoscopy
Annica K.B. Gad, Daniel Rönnlund, Jerker Widengren, and Pontus Aspenström
23. Yeast as a Model for Ras Signaling
Renata Tisi, Fiorella Belotti, and Enzo Martegani
24. Methods to Study the Ras2 Protein Activation State and the Subcellular Localization of Ras-GTP in Saccharomyces cerevisiae
Sonia Colombo and Enzo Martegani
25. RAS Proteins Signaling in the Early Metazoan Dictyostelium discoideum
Enrico Bracco and Barbara Pergolizzi
Klappentext
Featuring experimental approaches that shed light on the complexity of Ras GTPase biological functions, Ras Signaling: Methods and Protocols contains general overviews and detailed applications of both well-established and recently developed research techniques, including biochemical, biophysical, molecular biology, genetic and behavioral approaches, advanced high resolution fluorescence and electron microscopy imaging and ¿omics¿ technologies. Through this, the detailed volume provides information on expression, post-translational modifications, subcellular localization and dynamics, regulatory mechanisms of upstream and downstream signaling pathways and ultimately, biological activities and functions of Ras GTPases in different model systems, including high and low eukaryotic organisms. Written in the highly successful Methods in Molecular Biology series format, chapters include brief introductions, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips on troubleshooting and avoiding known pitfalls.nWide-ranging and authoritative, Ras Signaling: Methods and Protocols serves as an aid for investigators of different backgrounds and interests related to the multiple physiological and pathological functions of the large superfamily of Ras GTPases.
Includes experimental approaches to the complexity of the biological functions of RasGTPases
Provides step-by-step detail essential for reproducible results
Contains key notes and implementation advice from the experts
Includes supplementary material: sn.pub/extras
