Ras signaling : methods and protocols

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 -- Luca Goitre [and three others] A novel method for the production of fully modified K-Ras 4B -- Tanmay S. Chavan [and four others] Metabolic labeling of Ras with tritiated palmitate to monitor palmitoylation and depalmitoylation -- Frederick D. Tsai [and three others] Ubiquitination of Rac1 by inhibitors of apoptosis (IAPs) -- Tripat Kaur Oberoi-Khanuja and Krishnaraj Rajalingam Ras GTPases are both regulators and effectors of redox agents -- Elisa Ferro [and four others] Biophysical and proteomic characterization strategies for cysteine modifications in Ras GTPases -- G. Aaron Hobbs, Harsha P. Gunawardena, and Sharon L. Campbell Use of the yeast two-hybrid technology to isolate molecular interactions of Ras GTPases -- Elisa Ferro, Eva Baldini, and Lorenza Trabalzini Screening for MAPK modulators using an In-Cell Western assay -- Simon Schnaiter [and seven others] 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 Functional phosphoproteomics of oncogenic KRAS signaling -- Putty-Reddy Sudhir and Jeou-Yuan Chen Pull-down assay for analysis of integrin-mediated activation of Rap proteins in adherent platelets -- Gianni Francesco Guidetti and Mauro Torti Combined pulldown and time-lapse microscopy studies for determining the role of Rap1 in the crosstalk between integrins and cadherins -- Luca Goitre and Saverio Francesco Retta Fluorescence microscopy study of Rap1 subcellular localization -- Luca Goitre, Valentina Cutano, and Saverio Francesco Retta An in vitro system to evaluate the scaffold function of the RalA effector protein RalBP1 -- David F. Kashatus -- modulators using an In-Cell Western assay /-- Simon Schnaiter [and seven others] Analysis of the Rit subfamily GTPase-mediated signaling and neuronal differentiation and survival -- Geng-Xian Shi and Catherine N. Kaminski 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 High-resolution scanning electron microscopy for the imaging of nuclear pore complexes and Ran-mediated transport -- Lihi Shaulov, Boris Fichtman, and Amnon Harel Effector recruitment method to study spatially regulated activation of Ras and Rho GTPases -- Lauren P. Huff, Molly J. DeCristo, and Adrienne D. Cox Real-time visualization and quantification of native Ras activation in single living cells -- Christoph Biskup and Ignacio Rubio Nanoclustering and heterogeneous membrane diffusion of Ras studied by FRAP and RICS analysis -- Camilo Guzmán, Maja Šolman, and Daniel Abankwa --n assay /-- Simon Schnaiter [and seven others] 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 Analysis of Rho GTPase-induced localization of nanoscale adhesions using fluorescence nanoscopy -- Annica K.B. Gad [and three others] Yeast as a model for Ras signalling -- Renata Tisi, Fiorella Belotti, and Enzo Martegani Methods to study the Ras2 protein activation state and the subcellular localization of Ras-GTP in Saccharomyces cerevisiae -- Sonia Colombo and Enzo Martegani Ras proteins signaling in the early metazoan Dictyostelium discoideum -- Enrico Bracco and Barbara Pergolizzi.