Hedgehog Signaling Protocols

ISBN-13: 9781493949229 / Angielski / Miękka / 2016 / 200 str.

Natalia A. Riobo

Opis

The second edition of this volume provides novel protocols for the study of newly discovered function and modifications of components of the Hh pathways. The first few chapters discuss methods for stable delivery of soluble Hh proteins in cell cultures that can be adapted for in vivo studies, and mathematical models for studying Hh proteins gradient formation in tissues. The next few chapters explore studies in cell culture using fibroblasts that respond readily to Hh proteins. The book goes on to present protocols for evaluation of transcriptional activity of the Gli transcription factors in cell cultures and determine the expression level of endogenous Gli proteins. The final chapters talk about metabolic effects of noncanonical Hh signaling. 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 authoritative, Hedgehog Signaling Protocols, Second Edition presents researchers with a comprehensive understanding of the Hh pathway and its function in development and diseases.

Spis treści

1. Controlled Delivery of Sonic Hedgehog with a Heparin-Based Coacervate
Noah Ray Johnson and Yadong Wang

2. In Vivo Imaging of Hedgehog Transport in Drosophila Epithelia
Irene Seijo, Isabel Guerrero, and Marcus Bischoff

3. Modeling Hedgehog Signaling Through Flux-Saturated Mechanism
Óscar Sánchez, Juan Calvo, Carmen Ibáñez, Isabel Guerrero, and Juan Soler  

4. Evaluating the Activity of Smoothened Toward G Proteins Using [³⁵S] Guanosine 5’-(3-O-thio)triphosphate ([³⁵S]GTPγS)
David R. Manning, Feng Shen, and Natalia A. Riobo

5. Analysis of Smoothened Phosphorylation and Activation in Cultured Cells and Wing Discs of Drosophila
Kai Jiang and Jianhang Jia

6. Investigation of Protein-Protein Interactions and Conformational Changes in Hedgehog Signaling Pathway by FRET
Lin Fu, Xiangdong Lv, Yue Xiong, and Yun Zhao  

7. Luciferase Reporter Assays to Study Transcriptional Activity of HH Signaling in Normal and Cancer Cells
Silvia Pandolfi and Barbara Stecca

8. Measuring Expression Levels of Endogenous Gli Genes by Immunoblotting and Real-Time PCR
Pawel Niewiadomski and Rajat Rohatgi

9. Quantitative Immunoblotting of Endogenous Hedgehog Pathway Components
Shohreh F. Farzan and David J. Robbins

10. Measuring Gli2 Phosphorylation by Selected Reaction Monitoring Mass Spectrometry
Robert Ahrends, Pawel Niewiadomski, Mary N. Teruel, and Rajat Rohatgi

11. Rapid Screening of Gli2/3 Mutants Using the Flp-In System
Pawel Niewiadomski and Rajat Rohatgi

12. Insights into Gli Factors Ubiquitylation Methods
Paola Infante, Romina Alfonsi, and Lucia Di Marcotullio

13. Determination of Acetylation of the Gli Transcription Factors
Sonia Coni, Laura Di Magno, and Gianluca Canettieri

14. Efficient Detection of Indian Hedgehog During Endochondral Ossification by Whole Mount Immunofluorescence
João Francisco Botelho, Daniel Smith-Paredes, and Verónica Palma A.

15. Methods for Detection of Ptc1-Driven LacZ Expression in Adult Mouse SkinDonna M. Brennan-Crispi, Mỹ G. Mahoney, and Natalia A. Riobo

16. Determination and Analysis of Cellular Metabolic Changes by Non-Canonical Hedgehog Signaling
Raffaelle Teperino and John Andrew Pospisilik