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Methods Mol Biol. 2016;1481:17-28. doi: 10.1007/978-1-4939-6393-5_3.

Biochemical Methods to Analyze Wnt Protein Secretion.

Author information

1
Division Signaling and Functional Genomics and Heidelberg University, Department for Cell and Molecular Biology, German Cancer Research Center (DKFZ), Medical Faculty Mannheim, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.
2
Haematology and Oncology and Developmental Biochemistry, University Medicine Göttingen, Göttingen Center for Molecular Biosciences (GZMB), Justus-von-Liebig Weg 11, 37077, Göttingen, Germany. Julia.gross@med.uni-goettingen.de.

Abstract

Wnt proteins act as potent morphogens in various aspects of embryonic development and adult tissue homeostasis. However, in addition to its physiological importance, aberrant Wnt signaling has been linked to the onset and progression of different types of cancer. On the cellular level, the secretion of Wnt proteins involves trafficking of lipid-modified Wnts from the endoplasmic reticulum (ER) to Golgi and further compartments via the Wnt cargo receptor evenness interrupted. Others and we have recently shown that Wnt proteins are secreted on extracellular vesicles (EVs) such as microvesicles and exosomes. Although more details about specific regulation of Wnt secretion steps are emerging, it remains largely unknown how Wnt proteins are channeled into different release pathways such as lipoprotein particles, EVs and cytonemes. Here, we describe protocols to purify and quantify Wnts from the supernatant of cells by either assessing total Wnt proteins in the supernatant or monitoring Wnt proteins on EVs. Purified Wnts from the supernatant as well as total cellular protein content can be investigated by immunoblotting. Additionally, the relative activity of canonical Wnts in the supernatant can be assessed by a dual-luciferase Wnt reporter assay. Quantifying the amount of secreted Wnt proteins and their activity in the supernatant of cells allows the investigation of intracellular trafficking events that regulate Wnt secretion and the role of extracellular modulators of Wnt spreading.

KEYWORDS:

Blue Sepharose; Hydrophobic proteins; Purified Wnt; Wnt secretion; Wnt signaling activity; Wnts on extracellular vesicles

PMID:
27590148
DOI:
10.1007/978-1-4939-6393-5_3
[Indexed for MEDLINE]

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