FOXO Transcription Factors Methods and Protocols

This second edition volume expands on the previous edition with updates on the latest techniques and technologies used in FOXO research. The chapters in this book are organized into five parts. Part One covers biochemical techniques used to analyze phosphorylation status of FOXO proteins, identify FOXO binding partners, and assess FOXO-specific transcriptional activity. Part Two highlights genetic techniques that are used to identify functional domains and validate disease relevance. Part Three talks about biophysical and bioinformatic methods to study the structure and dynamics of FOXO proteins, and protein-protein interactions. Part Four discusses different fluorescence and imaging techniques to measure FOXO translocation, and Part Five focuses on functional studies in model organisms to characterize cell death regulation and life- and healthspan. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Comprehensive and cutting-edge, FOXO Transcription Factors: Methods and Protocols, Second Edition is a valuable resource for both expert and novel researchers who want to learn more about this exciting and developing field of FOXO biology.

FOXO Transcription Factors: A Brief Overview.- Phosphorylation of FOXO Proteins as a Key Mechanism to Regulate Their Activity.- Identification of DAF-16/FOXO Binding Partners in Caenorhabditis elegans by Immunoprecipitation and Mass Spectrometry.- Measuring FOXO Activity by Using qPCR-Based Expression Analysis of FOXO Target Genes.-   Reporter Gene Assays to Measure FOXO-Specific Transcriptional Activity.- Functional Motif Discovery in FOXO1 through CRISPR/Cas9 Exon Tiling Scan.- Cardiomyocyte-Specific FoxO1 Knockout Mice as Tools to Assess Cardiac Hypertrophy and Key Experimental Considerations using Cre-loxP.- Methods to Study Structure and Dynamics of FOXO Proteins.- Fluorescence Polarization Method to Assess the FOXO Phosphopeptide Interaction with 14-3-3.- Characterization of FOXO3-14-3-3 Interaction by Isothermal Titration Calorimetry.- Identification of Small-Molecule Modulators of FOXO3 through Virtual Screening.- Measuring FOXO Nuclear Shuttling Dynamics by Fluorescence Microscopy.- Fluorescent Tagging of Endogenous FOXO for Live Imaging and Pull-Down Assays.- Use of Microfluidics for Study of FOXO3 Translocation Dynamics.- Multiplexed Dual-Color Fluorescence-Based Distinction between Nuclear Trapping and Translocation of FOXO3.- Immunocytochemistry-Based Detection of FOXO Isoforms in Human Cancer and Fibroblasts.- Cell Death Regulation by Smed-foxO in the Planarian Schmidtea mediterranea.- Characterizing the Role of daf-16/ C. elegans FOXO in Lifespan and Healthspan.