Transforming Growth Factor-Beta Protocols (Methods in Molecular Biology Vol 142)

189 Pages·2000·2.22 MB·English

by  Philip H. Howe (Editor)

TM
Methods in Molecular Biology
VOLUME 142
Transforming
Growth
Factor-Beta
Protocols
Edited by
Philip H. Howe
HUMANA PRESS
Transforming Growth Factor-Beta Protocols
M E T H O D S I N M O L E C U L A R B I O L O G Y™
Transforming Growth
Factor-Beta Protocols
Edited by
Philip H. Howe
The Cleveland Clinic Foundation, Cleveland, OH
Humana Press Totowa, New Jersey
© 2000 Humana Press Inc.
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Preface
Since its original identification and isolation nearly 20 years ago, trans-
forming growth factor-beta (TGF`) has been extensively investigated with
respect to its ability to induce and modulate numerous physiological responses
in vitro and in vivo. However, attempts to analyze and identify the signal trans-
duction pathway(s) utilized by TGF` were, for the most part, fruitless and
remained elusive until the last decade when the molecule began to emerge
from its black box.
The isolation and identification of the TGF` receptors, as well as the
intracellular mediatory SMAD proteins, have allowed for the biochemical
characerization of at least some of the pathways involved in TGF` signaling.
As a result, many new methodologies and techniques are applicable to the
study of TGF` that until recently were a luxury reserved only for other growth
factors and cytokines. It is our aim in Transforming Growth Factor-Beta Pro-
tocols to encompass both old and new methodologies into a concise, self-
contained volume allowing both experts and novices a benchside reference.
The authors contributing the various chapters are all experts in their field
and they routinely perform the methodologies described. I thank them for their
time and effort. We hope that this protocol manual will become torn and tat-
tered from its frequent use on laboratory bench tops.
Philip H. Howe, PhD
v
Contents
Preface .............................................................................................................v
Contributors ..................................................................................................... ix
1 In Vitro Assays for Measuring TGF` Growth Stimulation
and Inhibition
Maryanne Edens and Edward B. Leof ................................................ 1
2 Measurements of Active TGF` Generated by Culture Cells
Roberta Mazzieri, John S. Munger, and Daniel B. Rifkin .............. 13
3 Methods for Iodination of Active TGF`, TGF` Receptor
Crosslinking and Immunoprecipitation of TGF`<Receptor
Complexes
David Danielpour ................................................................................ 29
4 Detection of TGF` in Body Fluids and Tissues
Andrew H. Limper ............................................................................... 39
5 Analysis of TGF`-Mediated Synthesis of Extracellular Matrix
Components
Barbara A. Hocevar and Philip H. Howe .......................................... 55
6 Characterization of SMAD Phosphorylation and SMAD/Receptor
Interaction
Arun Mehra, Liliana Attisano, and Jeffrey L. Wrana ...................... 67
7 Promoter Analysis of TGF`-Responsive Genes by Transient
Transfection and Deletional/Mutational Analysis
Ge Jin and Philip H. Howe ................................................................. 79
8 Regulation of AP-1 Activity by TGF`
Barbara A. Hocevar and Philip H. Howe .......................................... 97
9 CDK Pathway: Cyclin-Dependent Kinases (cdks) and
Cyclin-Dependent Kinase Inhibitors
Diana M. Gitig and Andrew Koff ..................................................... 109
10 Activation of the Mitogen-Activated Protein Kinase Pathway
by TGF`
Jianbo Yue and Sanford Markowitz ............................................... 125
11 Detection of TGF` Type II Receptor Hotspot Mutations:
The BAT-RII Assay
Lois L. Myeroff and Sanford Markowitz ......................................... 133
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viii Contents
12 Mutation Detection in the TGF` Receptors and SMAD Genes:
RT-PCR and Sequencing
Lois L. Myeroff, Hongmei He, Stephen P. Fink, and
Sanford Markowitz ....................................................................... 139
13 Analysis of TGF`-Inducible Apoptosis
Thomas L. Brown, Supriya Patil, and Philip H. Howe .................. 149
Index ......................................................................................................... 169
Contributors
LILIANA ATTISANO • Department of Anatomy and Cell Biology, University
of Toronto, Toronto, ON, Canada
THOMAS L. BROWN • Department of Cell Biology, Lerner Research Institute,
The Cleveland Clinic Foundation, Cleveland, OH
DAVID DANIELPOUR • Ireland Cancer Center of the University Hospitals
of Cleveland and Department of Pharmacology, Case Western Reserve
University, Cleveland, OH
MARYANNE EDENS • Thoracic Diseases Research Unit and Department
of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN
STEPHEN P. FINK • Howard Hughes Medical Institute, Cleveland, OH
DIANA M. GITIG • Program in Cell Biology and Genetics, Memorial Sloan-
Kettering Cancer Center, New York, NY; and Department of Molecular
Biology, Cornell University Graduate School of Medical Sciences,
Ithaca, NY
HONGMEI HE • Ireland Cancer Center, Case Western Reserve University,
Cleveland, OH
BARBARA A. HOCEVAR • Department of Cell Biology, Lerner Research
Institute, The Cleveland Clinic Foundation, Cleveland, OH
PHILIP H. HOWE • Department of Cell Biology, Lerner Research Institute,
The Cleveland Clinic Foundation, Cleveland, OH
GE JIN • Department of Cell Biology, Lerner Research Institute, The
Cleveland Clinic Foundation, Cleveland, OH
ANDREW KOFF • Department of Molecular Biology, Cornell University
Graduate School of Medical Sciences, Ithaca, NY; and Memorial Sloan-
Kettering Cancer Center, New York, NY
EDWARD B. LEOF • Thoracic Diseases Research Unit and Department of
Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN
ANDREW H. LIMPER • Department of Medicine, Biochemistry and Molecular
Biology, Mayo Clinic, Rochester, MN
SANFORD MARKOWITZ • Ireland Cancer Center, Case Western Reserve
University; and Howard Hughes Medical Institute, Cleveland, OH
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