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J Histochem Cytochem. 2014 Dec;62(12):846-63. doi: 10.1369/0022155414550163. Epub 2014 Aug 20.

Brightfield proximity ligation assay reveals both canonical and mixed transforming growth factor-β/bone morphogenetic protein Smad signaling complexes in tissue sections.

Author information

1
Laboratory of Cancer Biology and Genetics (KCF, BT, MS, SLD, LMW), Center for Cancer Research, National Cancer Institute, Bethesda, MDAntibody and Protein Purification Unit (CDH, PKG), Center for Cancer Research, National Cancer Institute, Bethesda, MDLaboratory of Pathology (CC, SMH), Center for Cancer Research, National Cancer Institute, Bethesda, MD flanderk@mail.nih.gov.
2
Laboratory of Cancer Biology and Genetics (KCF, BT, MS, SLD, LMW), Center for Cancer Research, National Cancer Institute, Bethesda, MDAntibody and Protein Purification Unit (CDH, PKG), Center for Cancer Research, National Cancer Institute, Bethesda, MDLaboratory of Pathology (CC, SMH), Center for Cancer Research, National Cancer Institute, Bethesda, MD.

Abstract

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.

KEYWORDS:

Smads; breast cancer; brightfield; development; proximity ligation assay; tissue microarray; transforming growth factor-β

PMID:
25141865
PMCID:
PMC4244299
DOI:
10.1369/0022155414550163
[Indexed for MEDLINE]
Free PMC Article

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