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This volume arranged into three sections describes biochemical, in vitro , and in vivo protocols on Semaphorins. Chapters focus on approaches that would allow the novice to study Semaphorins and employ robust assays to characterize mechanisms of action. 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.

Authoritative and cutting-edge, Semaphorin Signaling: Methods and Protocols aims to ensure successful results in the further study of this vital field.

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1. Semaphorins and their Signaling Mechanisms

Laura T. Alto and Jonathan R. Terman

Part I: Biochemical Approaches

2. Isolating Fc-Tagged SEMA4D Recombinant Protein from 293FT Cells

Joanne Soong and Glynis A. Scott

3. Expression and Purification of Class 7 Semaphorin and its Plexinc1 Receptor Using Baculovirus-Mediated Mammalian Cell Gene Transduction

Xiaoyan Chen, Po-Han Chen, and Xiaolin He

4. Immunoaffinity Purification of the Glycosylated Extracellular Fragment of Mouse Plexin A2 Produced in a Mammalian Expression System

Terukazu Nogi, Emiko Mihara, Norihisa Yasui, and Junichi Takagi

5. Plate-Based Assay for Measuring Direct Semaphorin-Neuropilin Interactions

Matthew W. Parker and Craig W. Vander Kooi

6. Characterizing Plexin Gtpase Interactions Using Gel Filtration, Surface Plasmon Resonance Spectrometry and Isothermal Titration Calorimetry

Jeannine Muller-Greven, Soonjeung Kim, Prasanta K. Hota, Yufeng Tong, Susmita Borthakur, and Matthias Buck

7. In Vitro Assay for the Rap Gtpase Activating Protein Activity of the Purified Cytoplasmic Domain of Plexin

Heath G. Pascoe, Yuxiao Wang, and Xuewu Zhang

8. Characterizing F-Actin Disassembly Induced by the Semaphorin Signaling Component Mical
Jimok Yoon, Ruei-Jiun Hung, and Jonathan R. Terman

9. Characterizing Erbb-2-Mediated Tyrosine Phosphorylation and Activation of Plexins

Thomas Worzfeld and Jakub M. Swiercz

10. Characterizing PKA-Mediated Phosphorylation of Plexin Using Purified Proteins

Taehong Yang and Jonathan R. Terman

Part II: In Vitro Approaches

11. Using Heterologous Cos-7 Cells to Identify Semaphorin Signaling Components Atsuko Sakurai, Colleen L. Do‡i, and J. Silvio Gutkind

12. Analysis of Semaphorin-Induced Growth Cone Collapse and Axon Growth Inhibition

Lionel Meyer, Andrius Kaselis, Saulius Satkauskas, and Dominique Bagnard

13. Using Rotary Shadow Electron Microscopy to Characterize Semaphorin-Mediated Growth Cone Collapse

Paul C. Bridgman

14. An Electrical Impedance-Based Method for Quantitative Real-Time Analysis of Semaphorin-Elicited Endothelial Cell Collapse

Chiara Camillo, Noemi Gioelli, Federico Bussolin, and Guido Serini

15. Regulation of Cortical Dendrite Morphology and Spine Organization by Secreted Semaphorins - A Primary Culture Approach

Sheng-Shiang Peng and Tracy S. Tran

16. Characterizing Semaphorin Signaling Using Isolated Neurons in Culture

Sophie Chauvet, Erik Mire, and Fanny Mann

17. Performing Axon Orientation Assays With Secreted Semaphorins And Other Guidance Cues

Céline Delloye-Bourgeois, Frédéric Moret And Valérie Castellani

18. Assays to Examine Transmembrane Semaphorin Function In Vitro

Rachel Q. Liu, Kristen Browne, and Timothy P. O´Connor

19. Micro-CALI to Study Localized Roles of the Semaphorin Signaling Component CRMP in Axon Growth

Yoshio Goshima and Kohtaro Takei

20. Visualizing and Characterizing Semaphorin Endocytic Events Using Quantum Dot Conjugated Proteins

Ioana Carcea and Deanna L. Benson

21. Visualization of Clathrin-Mediated Endocytosis during Semaphorin-Guided Axonal Growth

Rurika Itofusa, Takuro Tojima, and Hiroyuki Kamiguchi

22. Tracking Differential Endocytosis and Trafficking of Semaphorin Receptor Complexes in Responding Nerve Growth Cones

Puneet Dang and Andrew J. W. Furley

23. Antibody-Feeding Assay: A Method to Track the Internalization of Neuropilin-1 and Other Cell Surface Receptors

Sabrina Rizzolio and Luca Tamagnone

24. Photolithography-Based Substrate Microfabrication for Patterning Semaphorin 3A to Study Neuronal Development

Maya Shelly, Seong-Il Lee, Giulia Suarato, Yizhi Meng, and Sophie Pautot

25. Characterization of Semaphorin 6A-Mediated Effects on Angiogenesis through Regulation of VEGF Signaling

Hidetaka Ohnuki and Giovanna Tosato

26. Studying the Effects of Semaphorins on Oligodendrocyte Lineage Cells

Yasir Ahmed Syed Sarah Ali Abdulla, and

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