Novel association of APC with intermediate filaments identified using a new versatile APC antibody.
BMC Cell Biol
; 10: 75, 2009 Oct 21.
Article
en En
| MEDLINE
| ID: mdl-19845967
ABSTRACT
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 beta-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.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Poliposis Adenomatosa del Colon
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Proteínas de Filamentos Intermediarios
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Anticuerpos
Tipo de estudio:
Risk_factors_studies
Límite:
Humans
Idioma:
En
Revista:
BMC Cell Biol
Año:
2009
Tipo del documento:
Article
País de afiliación:
Estados Unidos