TGF-β Signaling: Methods and Protocols by Xin-Hua Feng, Pinglong Xu, Xia Lin

By Xin-Hua Feng, Pinglong Xu, Xia Lin

This quantity info updated details and makes an attempt to take the reader into the fascinating realm of TGF-β from the fundamental ideas to the sensible functions. Chapters offer simple creation of TGF-β signaling from the mobile floor to the nucleus, tools and strategies for the research of TGF-β signaling mechanism together with receptors, Smads, intracellular kinases, microRNA, epigenetic law, post-translational rules, non-Smad pathway; the physiological implications together with these in telephone cycle arrest, epithelial-mesenchymal transition, endothelial cells, adipogenesis, Th differentiation, stem telephone, bone home improvement, ovary, zebrofish improvement, and frog animal capping; and the methodologies together with metastasis imaging, 3D morphogenesis, membrane receptor quantification, conditional knockout, bone home improvement, kinase and phosphatase assays, BiFC interplay assays, and genome-wide siRNA reveal. Written within the hugely winning Methods in Molecular Biology sequence structure, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, without problems reproducible laboratory protocols, and key pointers on troubleshooting and warding off recognized pitfalls.

 

Authoritative and useful, TGF-Beta Signaling: equipment and Protocols goals to make sure winning ends up in the additional learn of this important field.

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TGF-β Signaling: Methods and Protocols

This quantity information up to date info and makes an attempt to take the reader into the fascinating realm of TGF-β from the elemental rules to the sensible purposes. Chapters offer simple creation of TGF-β signaling from the mobilephone floor to the nucleus, tools and strategies for the research of TGF-β signaling mechanism together with receptors, Smads, intracellular kinases, microRNA, epigenetic law, post-translational laws, non-Smad pathway; the physiological implications together with these in phone cycle arrest, epithelial-mesenchymal transition, endothelial cells, adipogenesis, Th differentiation, stem mobilephone, bone home improvement, ovary, zebrofish improvement, and frog animal capping; and the methodologies together with metastasis imaging, 3D morphogenesis, membrane receptor quantification, conditional knockout, bone home improvement, kinase and phosphatase assays, BiFC interplay assays, and genome-wide siRNA monitor.

Extra resources for TGF-β Signaling: Methods and Protocols

Example text

Lopez-Casillas F, Payne HM, Andres JL, Massague J (1994) Betaglycan can act as a dual modulator of TGF-beta access to signaling receptors: mapping of ligand binding and GAG attachment sites. J Cell Biol 124(4): 557–568 61. Guerrero-Esteo M, Sanchez-Elsner T, Letamendia A, Bernabeu C (2002) Extracellular and cytoplasmic domains of endoglin interact with the transforming growth factor-beta receptors I and II. J Biol Chem 277(32):29197–29209 62. Barbara NP, Wrana JL, Letarte M (1999) Endoglin is an accessory protein that interacts with the signaling receptor complex of multiple members of the transforming growth factor-beta superfamily.

Add 50 μL of protein A or G beads (50 % slurry, washed with solubilization buffer) and incubate for 45 min (rotate). 21. Wash the beads four to five times with solubilization buffer. TGF-β Receptors in Cell Membrane 41 22. Remove as much as possible of the supernatant and add 50 μL of 2× SDS sample buffer. 23. Boil samples for 5 min and run the samples on a SDS-PAGE gel (see Note 10). 24. After running the gel fix the gel for 30 min in fixation buffer at RT (shaking). 25. Incubate the gel for 45 min in drying buffer at RT (shaking).

7 % PFA for 15 min at room temperature with gentle rocking. 4. Wash cells three times for 5 min each in PBS-IHC. Permeabilization If the primary antibody recognizes an extracellular epitope of your protein then permeabilization may not be necessary to detect cell surface protein. Check your antibody data sheet before starting the experiment. Permeabilization is required to detect target protein distributed intracellularly. The process provides access to intracellular or organellar epitopes that might be otherwise inaccessible.

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