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First published online December 17, 2008
doi: 10.1242/10.1242/jcs.043927

Journal of Cell Science 122, 21-24 (2009)
Published by The Company of Biologists 2009
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Protein p0071 – an armadillo plaque protein that characterizes a specific subtype of adherens junctions

Ilse Hofmann1,2,*, Tanja Schlechter1,2,4, Cäcilia Kuhn3, Michaela Hergt3 and Werner W. Franke3

1 Joint Research Division Vascular Biology, Medical Faculty Mannheim, University of Heidelberg, Ludolf-Krehl-Street 13-17, 68167 Mannheim, Germany
2 The German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
3 Helmholtz Group for Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 581, 69120 Heidelberg, Germany
4 Progen Biotechnik, Maass-Street 30, 69123 Heidelberg, Germany


Figure 1
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  		Fig. 1. (A) Schematic presentation of the domain structure of protein p0071 and the position of the amino acid (aa) sequences selected for the synthesis of oligopeptides used for immunization. (B,C) Detection of protein p0071 in (B) total lysates of human, (C, left panel) total lysates of rodent cell lines, and (C, right panel) total extracts of murine tissues. Cell lysates of the human cell lines HaCaT, MCF-7, A431, CaCo-2, PLC, 16 HBE14o, SV80 and glioma U333 (B) were processed for SDS-PAGE, and western blotted using mAb clone p0071-2. Notice the reactive polypeptide bands at ~135 kDa (all lanes) that can also be seen in the western blot analysis of the rodent cell line cultures MH1C1 (rat liver hepatocytes), 3T3 (mouse embryonal mesenchyme) and RVF-SM. (D) Immunoprecipitation experiments using total MCF-7 cell lysates and antibodies specific for protein p0071. Lanes 1, total cell lysates; lanes 2, negative controls for unspecific binding; lanes 3, immunoprecipitates. Western blotting using antibodies against (a) p0071, (b) desmoglein 1 and 2 (Dsg), (c) E-cadherin (E-cad) and (d) β-catenin (β-cat). Notice that in lanes 3, anti-p0071-1 immunoprecipitates were seen as an intense reaction for p0071 (a) and the complete absence of reaction for desmoglein (b); distinct amounts of p0071 were found in complexes of p0071 with E-cadherin (c) and β-catenin (d). Molecular mass markers are given at the left in kDa. (E) Immunoelectron microscopy image showing the localization of protein p0071 (immuno-gold particles enhanced by silver staining) to puncta adhaerentia in junctions (square brackets) of MCF-7 cells, and its absence from desmosomes (D) and bundles of intermediate-sized filaments. In panels A-E the following specific antibodies were used: mAb clone p0071-2 (B), guinea pig anti-mp0071 (C), mAb clone p0071-1 (D, a), mAb DG3.10 (D, b; Progen Biotechnik), monoclonal anti-E-cadherin (D, c; BD Biosciences Pharmingen), rabbit anti-β-catenin (D, d; from Sigma), guinea pig anti-hp0071 (E). Bar, 500 nm.

 

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  		Fig. 2. (A-D'') Double-labeling immunolocalization experiments using (A-A'') cultured cells or (B-D'') tissues. (A) Laser-scanning confocal immunofluorescence micrographs showing the results of a typical double-labeling experiment using a monolayer culture of human epithelial MCF-7 cells at high density. Immunolocalization of p0071 using mAb clone p0071-3 (red) is compared with that of demsoplakin (green). Notice that the small puncta adhaerentia that contain p0071 are distinct from the desmosomes that contain desmoplakin. (B,B') Localization of p0071 in the lactating bovine mammary gland. Confocal laser-scanning micrograph showing the immunolocalization of (B) p0071 (red; using mAb clone p0071-4) compared with that of (B') β-catenin (green). (B'') Merged image. Arrows denote a small blood capillary with an endothelium that stains positive for p0071. (C-D'') Localization of p0071 in (C-C'') rat and (D-D'') human hearts. Micrographs were taken using a laser-scanning confocal microscope; location of p0071 was established using mAb clone p0071-4 (red) was compared with that of (D') desmoplakin (green; DP) or N-cadherin (C', green; N-cad). (C'',D'') Merged color images. Bars, 50 µm (B-B'', C-C''), 20 µm (A, D-D'').

 

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© The Company of Biologists Ltd 2009