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Open Access Highly Accessed Research article

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

Yang Wang1, Yoshiaki Azuma1, David B Friedman2, Robert J Coffey3 and Kristi L Neufeld1*

Author Affiliations

1 Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA

2 Mass Spectrometry Research Center, Vanderbilt University Medical Center, Nashville, TN, USA

3 Departments of Cell and Developmental Biology and Medicine, Vanderbilt University Medical Center, Nashville, TN, USA

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BMC Cell Biology 2009, 10:75  doi:10.1186/1471-2121-10-75

Published: 21 October 2009

Additional files

Additional file 1:

Generation of recombinant M2-APC immunogen. (A) Schematic diagram of the N-terminal His and S dual-tag fused APC fragment (amino acid 1000-1326) which contains the three 15 amino acid repeats and one 20 amino acid repeat. (B) Purified recombinant M2-APC protein (~50 kDa) used for immunization was resolved by SDS-PAGE and detected using colloidal blue.

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Additional file 2:

APC-M2 pAb co-precipitates APC binding proteins. Proteins co-precipitated from HCT116βw cell lysates using APC-M2 pAb were resolved on a 4-12% NUPAGE gel followed by colloidal blue staining. Stars mark the 9 protein bands that were precipitated using the APC-M2 pAb and not using preimmune sera.

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Additional file 3:

Full list of proteins associated with APC as identified with LC-MS/MS. Table.

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Additional file 4:

Negative controls for immunofluorescent microscopy analysis of HCT116βw cells. (A) Cells processed for conventional immunofluorescent microscopy using either APC-M2 pAb or no primary antibody, followed by goat-anti-mouse Alexa 488 secondary antibody reveal no recognition of the purified rabbit sera by the goat-anti-mouse secondary antibody. (B) Cells processed for conventional immunofluorescent microscopy using mouse monoclonal antibody against lamin B1, pan-keratin, or APC (ali12-28) or no primary antibody, followed by goat-anti-rabbit Alexa 568 secondary antibody reveal no recognition of the mouse monoclonal antibodies by the goat-anti-rabbit secondary antibody. (C) Cells processed for confocal microscopy were stained with goat-anti-rabbit Alexa 568 and goat-anti-mouse Alexa 488 secondary antibodies without primary antibody application. These images served as the negative control for the correlation coefficient analysis. Scale bar, 5 μm.

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