Titlebook: Hedgehog Signaling; Methods and Protocol Xiaochun Li Book 2022 The Editor(s) (if applicable) and The Author(s), under exclusive license to

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Cell-Based Assays for Smoothened Ubiquitination and Sumoylation,d activity of the GPCR family protein Smoothened (Smo). Smo trafficking and subcellular accumulation are controlled by multiple posttranslational modifications (PTMs) including phosphorylation, ubiquitination, and sumoylation, which appears to be conserved from . to mammals. Smo ubiquitination is dy

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Studies of SMOOTHENED Activation in Cell-Free and Reconstituted Systems,of cell-free and reconstituted systems to study a key step in Hedgehog signal transduction: the activation of SMOOTHENED by membrane lipids. These methods can be adapted to study other steps in Hedgehog signal transduction, particularly those that occur at the membrane.

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Rapid, Direct SMOOTHENED Activity Assays in Live Cells Using cAMP-Based Conformational Sensors,e a real-time cell-based SMO functional assay based on SMO activity-dependent changes in cellular cAMP concentrations. This assay is capable of detecting changes in SMO conformation within minutes of PTCH1 inactivation by Hh ligands. As a result, it expands the range of experimental perturbations th

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Light-Induced Differentiation of Forebrain Organoids by NVOC-SAG,its ligands finely controls the activity of the Hedgehog pathway during development. To mimic the active regulation of Hedgehog pathway, we have developed a light-inducible Hedgehog signaling activator 6-nitroveratryloxy-carbonyl Smoothened agonist (NVOC-SAG). Here we describe a method to selectivel

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Measuring Smoothened (SMO)-Mediated Activation of the Gi Protein,nscription factors. Previous studies have shown that SMO is a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F) that can activate G. family of heterotrimeric G proteins. Here, we describe [.S]-GTPγS assay using SMO cell membranes and purified G. protein to measure the level of G. pro

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Carina J?gers,Henk Roelink. Accessory cells are known to collaborate with lymphocytes and enable them to carry out their essential functions. FDC present antigen (Ag) to B cells and facilitate B cell functions; they may be thought of as B cell-associated accessory cells. In contrast, other dendritic cells, including Langerha