In vitro assays for measuring TGF-[beta] growth stimulation and inhibition -- Measurement of active TGF-[beta] generated by cultured cells -- Iodination of active TGF-[beta], TGF-[beta]-receptor crosslinking, and immunoprecipitation of TGF-[beta]-receptor complexes -- Deterction of TGF-[beta] in body fluids and tissues -- Analysis of TGF-[beta]-mediated synthesis of extracellular matrix components -- Characterizatio of SMAD phosphorylatin and SMAD-receptor interaction -- Promoter analysis of TGF-[beta] responsive genes by transient transfection and deletional/mutational analysis -- Regulation of AP-1 activity by TGF-[beta] -- Cdk pathway: cyclin-dependent kinases and cyclin-dependent kinase inhibitors -- Activation of the mitogen-activated protein kinase pathway by transforming growth factor-[beta] -- Detection of TGF-[beta] type 11 receptor hot-spot mutations: the BAT-RII assay -- Mutation detection in the TGF-[beta] receptors and smad genes: RT-PCR and sequencing -- Analysis of TGF-[beta]-inducible apoptosis.
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The recent isolation and identification of the TGFß receptors, as well as their intracellular mediating proteins, has driven the development of many new methodologies and techniques for the study of TGFß. In Transforming Growth Factor-Beta Protocols, Philip H. Howe and a group of well-versed experimentalists present the first major collection of indispensable classic and cutting-edge TGFß assays. Described in great detail to ensure robust and successful results, these readily reproducible techniques range from the growth inhibition assay for TGFß to methodologies for monitoring its interactions with the mediating proteins. Extensive notes discuss potential pitfalls and provide tips on how to avoid failures, and throughout, emphasis is given to detailing those technical steps critical for experimental success that are often omitted in the primary literature. Concise and highly practical, Transforming Growth Factor-Beta Protocols provides today's molecular and cell biologists-both expert and novice-with time-tested methods for the identification and analysis of the signal transduction pathways by which TGFß induces and modulates physiological behavior.