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This detailed book explores the technical breakthroughs with biophysical and cell biological approaches that have advanced the study of molecular mechanisms underlying the generation and transduction of the hedgehog (HH) signal, indicating the important role of sterols in this pathway. Within its pages, the volume examines techniques involving several key protein components in this pathway, including HH, the signaling ligand; Dispatched, a HH secretion regulator; Patched, the receptor of HH; Smoothened, the signal transducer used to transduce the HH signal across the plasma membrane; and GLI, the transcription factor to turn on HH target genes. Written in the highly successful Methods in Molecular Biology series format, chapters feature introductions to their respective topics, lists of the necessary materials and reagents, step-by-step readily reproducible laboratory protocols, as well as tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Hedgehog Signaling: Methods and Protocols provides researchers in clinical and basic science with a fresh and reliable reference for analyzing HH signal transduction through cell biological, biochemical, and biophysical approaches.
List of contents
Generation and Analysis of Mosaic Spinal Cord Organoids Derived from Mouse Embryonic Stem Cells.- Evaluating Hedgehog Acyltransferase Activity and Inhibition Using the Acylation-Coupled Lipophilic Induction of Polarization (Acyl-cLIP) Assay.- Analysis of Protein Cholesterylation by Biorthogonal Labeling.- An In Situ Fluorescence Assay for Cholesterol Transporter Activity of the Patched.- Imaging of the Ciliary Cholesterol Underling the Sonic Hedgehog Signal Transduction.- Studying Hedgehog Signaling during Mouse Neural Tube Development.- Measuring and Manipulating Membrane Cholesterol for the Study of Hedgehog Signaling.- The Immunofluorescence-Based Detection of Hedgehog Pathway Components in Primary Cilia of Cultured Cells.- Analysis of Dispatched Protein Processing and Sonic Hedgehog Ligand Release.- Expression, Purification, and Structure Determination of Human PTCH1-HH-N Complexes.- Characterization of Smoothened Phosphorylation and Activation.- Cell-Based Assays for Smoothened Ubiquitination and Sumoylation.- Biochemical Assays to Directly Assess Smoothened Activation by a Conformationally Sensitive Nanobody.- Studies of SMOOTHENED Activation in Cell-Free and Reconstituted Systems.- Rapid, Direct SMOOTHENED Activity Assays in Live Cells Using cAMP-Based Conformational Sensors.- Light-Induced Differentiation of Forebrain Organoids by NVOC-SAG.- NanoBRET and NanoBiT/BRET-Based Ligand Binding Assays Permit Quantitative Assessment of Small Molecule Ligand Binding to Smoothened.- Measuring Smoothened (SMO)-Mediated Activation of the Gi Protein.- Ci/Gli Phosphorylation by the Fused/Ulk Family Kinases.- A Method for Orthotopic Transplantation of Lung Cancer in Mice.
