Research article
Novel association of APC with intermediate filaments identified using a new versatile APC antibody
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* Corresponding author: Kristi L Neufeld klneuf@ku.edu
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
BMC Cell Biology 2009, 10:75 doi:10.1186/1471-2121-10-75
Published: 21 October 2009Additional 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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