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Novel association of APC with intermediate filaments identified using a new versatile APC antibody

BACKGROUND: As a key player in suppression of colon tumorigenesis, Adenomatous Polyposis Coli (APC) has been widely studied to determine its cellular functions. However, inconsistencies of commercially available APC antibodies have limited the exploration of APC function. APC is implicated in spindl...

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Autores principales: Wang, Yang, Azuma, Yoshiaki, Friedman, David B, Coffey, Robert J, Neufeld, Kristi L
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774295/
https://www.ncbi.nlm.nih.gov/pubmed/19845967
http://dx.doi.org/10.1186/1471-2121-10-75
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author Wang, Yang
Azuma, Yoshiaki
Friedman, David B
Coffey, Robert J
Neufeld, Kristi L
author_facet Wang, Yang
Azuma, Yoshiaki
Friedman, David B
Coffey, Robert J
Neufeld, Kristi L
author_sort Wang, Yang
collection PubMed
description BACKGROUND: As a key player in suppression of colon tumorigenesis, Adenomatous Polyposis Coli (APC) has been widely studied to determine its cellular functions. However, inconsistencies of commercially available APC antibodies have limited the exploration of APC function. APC is implicated in spindle formation by direct interactions with tubulin and microtubule-binding protein EB1. APC also interacts with the actin cytoskeleton to regulate cell polarity. Until now, interaction of APC with the third cytoskeletal element, intermediate filaments, has remained unexamined. RESULTS: We generated an APC antibody (APC-M2 pAb) raised against the 15 amino acid repeat region, and verified its reliability in applications including immunoprecipitation, immunoblotting, and immunofluorescence in cultured cells and tissue. Utilizing this APC-M2 pAb, we immunoprecipitated endogenous APC and its binding proteins from colon epithelial cells expressing wild-type APC. Using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), we identified 42 proteins in complex with APC, including β-catenin and intermediate filament (IF) proteins lamin B1 and keratin 81. Association of lamin B1 with APC in cultured cells and human colonic tissue was verified by co-immunoprecipitation and colocalization. APC also colocalized with keratins and remained associated with IF proteins throughout a sequential extraction procedure. CONCLUSION: We introduce a versatile APC antibody that is useful for cell/tissue immunostaining, immunoblotting and immunoprecipitation. We also present evidence for interactions between APC and IFs, independent of actin filaments and microtubules. Our results suggest that APC associates with all three major components of the cytoskeleton, thus expanding potential roles for APC in the regulation of cytoskeletal integrity.
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spelling pubmed-27742952009-11-07 Novel association of APC with intermediate filaments identified using a new versatile APC antibody Wang, Yang Azuma, Yoshiaki Friedman, David B Coffey, Robert J Neufeld, Kristi L BMC Cell Biol Research Article BACKGROUND: As a key player in suppression of colon tumorigenesis, Adenomatous Polyposis Coli (APC) has been widely studied to determine its cellular functions. However, inconsistencies of commercially available APC antibodies have limited the exploration of APC function. APC is implicated in spindle formation by direct interactions with tubulin and microtubule-binding protein EB1. APC also interacts with the actin cytoskeleton to regulate cell polarity. Until now, interaction of APC with the third cytoskeletal element, intermediate filaments, has remained unexamined. RESULTS: We generated an APC antibody (APC-M2 pAb) raised against the 15 amino acid repeat region, and verified its reliability in applications including immunoprecipitation, immunoblotting, and immunofluorescence in cultured cells and tissue. Utilizing this APC-M2 pAb, we immunoprecipitated endogenous APC and its binding proteins from colon epithelial cells expressing wild-type APC. Using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), we identified 42 proteins in complex with APC, including β-catenin and intermediate filament (IF) proteins lamin B1 and keratin 81. Association of lamin B1 with APC in cultured cells and human colonic tissue was verified by co-immunoprecipitation and colocalization. APC also colocalized with keratins and remained associated with IF proteins throughout a sequential extraction procedure. CONCLUSION: We introduce a versatile APC antibody that is useful for cell/tissue immunostaining, immunoblotting and immunoprecipitation. We also present evidence for interactions between APC and IFs, independent of actin filaments and microtubules. Our results suggest that APC associates with all three major components of the cytoskeleton, thus expanding potential roles for APC in the regulation of cytoskeletal integrity. BioMed Central 2009-10-21 /pmc/articles/PMC2774295/ /pubmed/19845967 http://dx.doi.org/10.1186/1471-2121-10-75 Text en Copyright © 2009 Wang et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Wang, Yang
Azuma, Yoshiaki
Friedman, David B
Coffey, Robert J
Neufeld, Kristi L
Novel association of APC with intermediate filaments identified using a new versatile APC antibody
title Novel association of APC with intermediate filaments identified using a new versatile APC antibody
title_full Novel association of APC with intermediate filaments identified using a new versatile APC antibody
title_fullStr Novel association of APC with intermediate filaments identified using a new versatile APC antibody
title_full_unstemmed Novel association of APC with intermediate filaments identified using a new versatile APC antibody
title_short Novel association of APC with intermediate filaments identified using a new versatile APC antibody
title_sort novel association of apc with intermediate filaments identified using a new versatile apc antibody
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774295/
https://www.ncbi.nlm.nih.gov/pubmed/19845967
http://dx.doi.org/10.1186/1471-2121-10-75
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