Titlebook: CD95; Methods and Protocol Patrick Legembre Book 2017 Springer Science+Business Media LLC 2017 Cell motility.Immunoprecipitation.TNF Recept

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CD95-Mediated Calcium Signaling,on is an exquisite readout to explore the molecular mechanisms elicited by CD95 engagement. The most widely applied methods for studying calcium signaling pathways use fluorescent indicators and imaging methods with fluorescence microscopy. This technical approach, however, requires many precautions that we discuss in this chapter.

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Quantifying CD95/cl-CD95L Implications in Cell Mechanics and Membrane Tension by Atomic Force Microesent a methodology that allows to quantify cell elastic properties (using the Young modulus) and cell membrane tension modulated by CD95/cl-CD95L interactions by coupling nanoindentation and membrane tube pulling using suitably decorated AFM levers.

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Immunoprecipitation of Death Inducing Signaling Complex by Caspase-8,tic separation by western blotting or mass spectrometry. Herein, we describe in detail the method used in our laboratory to immunoprecipitate and analyze by immunoblot complexes containing caspase-8, using a commercial antibody directed against caspase-8.

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Fluorometric Methods for Detection of Mitochondrial Membrane Depolarization Induced by CD95 Activattablished protocol using classical fluorescent probes, DIOC.(3) and TMRM. Living cells are loaded with these cationic dyes, which accumulate in mitochondria. After CD95 activation, organelle depolarization is assessed using flow cytometry.

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Isolation of Lipid Rafts Through Discontinuous Sucrose Gradient Centrifugation and Fas/CD95 Death R of the lipid raft resistance to Triton X-100 solubilization at 4 °C, followed by sucrose gradient centrifugation, with subsequent analysis of Fas/CD95 death receptor localization in the raft fractions by immunoblotting. This method is also useful to localize additional proteins in membrane rafts.