Redefining the subcellular location and transport of APC: new insights using a panel of antibodies

Brocardo, Mariana G.; Näthke, Inke; Henderson, Beric R.

doi:10.1038/sj.embor.7400329

Cited by 57 publications

(77 citation statements)

References 28 publications

(61 reference statements)

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“…Here, we describe the detection specificities of a panel of antibodies that have been developed to detect APC. This work extends several recent publications in this area and importantly serves to provide a comprehensive analysis of APC antibody specificities (Roberts et al, 2003;Brocardo et al, 2005).…”

Section: Discussionsupporting

confidence: 71%

“…We report these findings and present a summary of the antibody specificities for the panel of commonly used antibodies to APC. This study supports and expands on the recent study by Brocardo et al (2005) and includes commonly used antibodies to APC that were not tested in their study. The relevance of reporting such antibody specificities lies in the central importance of APC to cancer development.…”

supporting

confidence: 87%

“…The staining patterns observed using these antibodies are not in accordance with the observations of Brocardo et al (2005). Furthermore, that the C-terminal APC antibodies should give the same staining pattern as the N-terminal antibodies to APC in SW480 cells was unusual, as C-terminal APC sequences should not be expressed in these cells.…”

Section: Resultscontrasting

confidence: 56%

“…After many conflicting reports, it has recently been shown (through a combination of Western blotting, siRNA and IF) that endogenous full-length and truncated APC is predominantly located in the cytoplasm (Brocardo et al, 2005). Our panel of antibodies included commonly used antibodies to APC that were not tested in the study by Brocardo and co-workers.…”

Section: Resultsmentioning

confidence: 99%

“…A recent study showed that both full-length and truncated APC are present in the cytoplasm and that previous contradicting reports had been the result of aberrant antibody specificities. (Brocardo et al, 2005). The latter report (amongst others) highlights the importance of confirming the detection specificities of antibodies to APC.…”

mentioning

confidence: 85%

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Analysis of a panel of antibodies to APC reveals consistent activity towards an unidentified protein

et al. 2007

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Acquisition of truncating mutations in the adenomatous polyposis coli (APC) protein underlies the progression of the majority of sporadic and familial colorectal cancers. As such, the localisation patterns and interacting partners of APC have been extensively studied in a range of systems, relying on the use of a broad panel of antibodies. Until recently, antibodies to APC have been used largely unchecked. However, several recent reports have been invaluable in clarifying the use of a number of antibodies commonly used to detect APC. Here, we analyse the specificity of a further subset of antibodies to APC. We used a panel of six commercially available antibodies (directed to the amino and carboxy termini of APC) and confirm the detection of full-length APC by immunoblotting. We demonstrate that a 150 kDa protein, also reproducibly detected by this panel of antibodies, is unlikely to be APC. We present data for the immunological staining patterns of the APC antibodies and validate the results through RNAi. Using this approach, we confirm that the apical staining pattern, observed by immunofluorescence and previously reported in cell systems, is unlikely to be APC. Finally, we present our data as a summary of APC-antibody specificities for APC.

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Section: Discussionsupporting

confidence: 71%

supporting

confidence: 87%

Section: Resultscontrasting

confidence: 56%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 85%

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Analysis of a panel of antibodies to APC reveals consistent activity towards an unidentified protein

et al. 2007

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Regulation of BRCA1, BRCA2 and BARD1 intracellular trafficking

Henderson

2005

BioEssays

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The subcellular location and function of many proteins are regulated by nuclear-cytoplasmic shuttling. BRCA1 and BARD1 provide an interesting model system for understanding the influence of protein dimerization on nuclear transport and localization. These proteins function predominantly in the nucleus to regulate cell cycle progression, DNA repair/recombination and gene transcription, and their export to the cytoplasm has been linked to apoptosis. Germ-line mutations in the BRCA1/BRCA2 and BARD1 genes predispose to risk of breast/ovarian cancer, and certain mutations impair protein function and nuclear accumulation. BRCA1 and BARD1 shuttle between the nucleus and cytoplasm; however heterodimerization masks the nuclear export signals located within each protein, causing nuclear retention of the BRCA1-BARD1 complex and potentially influencing its role in DNA repair, cell survival and regulation of centrosome duplication. This review discusses BRCA1, BRCA2 and BARD1 subcellular localization with emphasis on regulation of transport by protein dimerization and its functional implications.

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Purification and characterization of a high specificity polyclonal antibody to the adenomatous polyposis coli tumour suppressor protein

Catimel

Nice

Karrlander

et al. 2006

Biomedical Chromatography

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Recombinant proteins, commonly expressed in fusion with an affinity tag to facilitate purification, are often used as immunogens for polyclonal antibody production. Careful immunopurification of the antibody product is often the key to obtaining a high-specificity polyclonal antibody against the protein domain of interest. This study describes the purification and characterization of such an antibody directed against the adenomatous polyposis coli (APC) tumour suppressor. We used a combination of affinity chromatography and biosensor analysis to optimize and monitor antibody purification. This antibody was then characterized by immunoprecipitation, proteomic analyses and immunofluorescence staining and shown to be a valuable reagent for the study of APC biology. Using this antibody we successfully isolated and identified APC, using MS/MS, from transfected cell lines. A novel phosphorylation site on APC was identified at ser 1436. Similar strategies involving multiple immuno-affinity steps coupled with surface plasmon resonance (SPR), immunoprecipitation proteomic and immunofluorescence analyses should be generally applicable for the purification and characterization of other polyclonal antibodies.

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Redefining the subcellular location and transport of APC: new insights using a panel of antibodies

Cited by 57 publications

References 28 publications

Analysis of a panel of antibodies to APC reveals consistent activity towards an unidentified protein

Analysis of a panel of antibodies to APC reveals consistent activity towards an unidentified protein

Regulation of BRCA1, BRCA2 and BARD1 intracellular trafficking

Purification and characterization of a high specificity polyclonal antibody to the adenomatous polyposis coli tumour suppressor protein

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