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Brightfield Proximity Ligation Assay Reveals Both Canonical and Mixed Transforming Growth Factor-β/Bone Morphogenetic Protein Smad Signaling Complexes in Tissue Sections

Transforming growth factor-β (TGF-β) is an important regulator of cellular homeostasis and disease pathogenesis. Canonical TGF-β signaling occurs through Smad2/3–Smad4 complexes; however, recent in vitro studies suggest that elevated levels of TGF-β may activate a novel mixed Smad complex (Smad2/3-S...

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Autores principales: Flanders, Kathleen C., Heger, Christopher D., Conway, Catherine, Tang, Binwu, Sato, Misako, Dengler, Samuel L., Goldsmith, Paul K., Hewitt, Stephen M., Wakefield, Lalage M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: SAGE Publications 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4244299/
https://www.ncbi.nlm.nih.gov/pubmed/25141865
http://dx.doi.org/10.1369/0022155414550163
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author Flanders, Kathleen C.
Heger, Christopher D.
Conway, Catherine
Tang, Binwu
Sato, Misako
Dengler, Samuel L.
Goldsmith, Paul K.
Hewitt, Stephen M.
Wakefield, Lalage M.
author_facet Flanders, Kathleen C.
Heger, Christopher D.
Conway, Catherine
Tang, Binwu
Sato, Misako
Dengler, Samuel L.
Goldsmith, Paul K.
Hewitt, Stephen M.
Wakefield, Lalage M.
author_sort Flanders, Kathleen C.
collection PubMed
description Transforming growth factor-β (TGF-β) is an important regulator of cellular homeostasis and disease pathogenesis. Canonical TGF-β signaling occurs through Smad2/3–Smad4 complexes; however, recent in vitro studies suggest that elevated levels of TGF-β may activate a novel mixed Smad complex (Smad2/3-Smad1/5/9), which is required for some of the pro-oncogenic activities of TGF-β. To determine if mixed Smad complexes are evident in vivo, we developed antibodies that can be used with a proximity ligation assay to detect either canonical or mixed Smad complexes in formalin-fixed paraffin-embedded sections. We demonstrate high expression of mixed Smad complexes in the tissues from mice genetically engineered to express high levels of TGF-β1. Mixed Smad complexes were also prominent in 15–16 day gestation mouse embryos and in breast cancer xenografts, suggesting important roles in embryonic development and tumorigenesis. In contrast, mixed Smad complexes were expressed at extremely low levels in normal adult mouse tissue, where canonical complexes were correspondingly higher. We show that this methodology can be used in archival patient samples and tissue microarrays, and we have developed an algorithm to quantitate the brightfield read-out. These methods will allow quantitative analysis of cell type-specific Smad signaling pathways in physiological and pathological processes.
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spelling pubmed-42442992015-12-01 Brightfield Proximity Ligation Assay Reveals Both Canonical and Mixed Transforming Growth Factor-β/Bone Morphogenetic Protein Smad Signaling Complexes in Tissue Sections Flanders, Kathleen C. Heger, Christopher D. Conway, Catherine Tang, Binwu Sato, Misako Dengler, Samuel L. Goldsmith, Paul K. Hewitt, Stephen M. Wakefield, Lalage M. J Histochem Cytochem Articles Transforming growth factor-β (TGF-β) is an important regulator of cellular homeostasis and disease pathogenesis. Canonical TGF-β signaling occurs through Smad2/3–Smad4 complexes; however, recent in vitro studies suggest that elevated levels of TGF-β may activate a novel mixed Smad complex (Smad2/3-Smad1/5/9), which is required for some of the pro-oncogenic activities of TGF-β. To determine if mixed Smad complexes are evident in vivo, we developed antibodies that can be used with a proximity ligation assay to detect either canonical or mixed Smad complexes in formalin-fixed paraffin-embedded sections. We demonstrate high expression of mixed Smad complexes in the tissues from mice genetically engineered to express high levels of TGF-β1. Mixed Smad complexes were also prominent in 15–16 day gestation mouse embryos and in breast cancer xenografts, suggesting important roles in embryonic development and tumorigenesis. In contrast, mixed Smad complexes were expressed at extremely low levels in normal adult mouse tissue, where canonical complexes were correspondingly higher. We show that this methodology can be used in archival patient samples and tissue microarrays, and we have developed an algorithm to quantitate the brightfield read-out. These methods will allow quantitative analysis of cell type-specific Smad signaling pathways in physiological and pathological processes. SAGE Publications 2014-12 /pmc/articles/PMC4244299/ /pubmed/25141865 http://dx.doi.org/10.1369/0022155414550163 Text en © The Author(s) 2014 http://creativecommons.org/licenses/by-nc/3.0/ This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (http://www.uk.sagepub.com/aboutus/openaccess.htm).
spellingShingle Articles
Flanders, Kathleen C.
Heger, Christopher D.
Conway, Catherine
Tang, Binwu
Sato, Misako
Dengler, Samuel L.
Goldsmith, Paul K.
Hewitt, Stephen M.
Wakefield, Lalage M.
Brightfield Proximity Ligation Assay Reveals Both Canonical and Mixed Transforming Growth Factor-β/Bone Morphogenetic Protein Smad Signaling Complexes in Tissue Sections
title Brightfield Proximity Ligation Assay Reveals Both Canonical and Mixed Transforming Growth Factor-β/Bone Morphogenetic Protein Smad Signaling Complexes in Tissue Sections
title_full Brightfield Proximity Ligation Assay Reveals Both Canonical and Mixed Transforming Growth Factor-β/Bone Morphogenetic Protein Smad Signaling Complexes in Tissue Sections
title_fullStr Brightfield Proximity Ligation Assay Reveals Both Canonical and Mixed Transforming Growth Factor-β/Bone Morphogenetic Protein Smad Signaling Complexes in Tissue Sections
title_full_unstemmed Brightfield Proximity Ligation Assay Reveals Both Canonical and Mixed Transforming Growth Factor-β/Bone Morphogenetic Protein Smad Signaling Complexes in Tissue Sections
title_short Brightfield Proximity Ligation Assay Reveals Both Canonical and Mixed Transforming Growth Factor-β/Bone Morphogenetic Protein Smad Signaling Complexes in Tissue Sections
title_sort brightfield proximity ligation assay reveals both canonical and mixed transforming growth factor-β/bone morphogenetic protein smad signaling complexes in tissue sections
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4244299/
https://www.ncbi.nlm.nih.gov/pubmed/25141865
http://dx.doi.org/10.1369/0022155414550163
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