title: Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay.
abstract: Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins. Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.
authors.affiliation: University Children's Hospital Basel (UKBB), University of Basel; xinzhou.zhu@ukbb.ch.
authors.forename: Xinzhou
authors.initials: X
authors.lastname: Zhu
authors.affiliation: University Children's Hospital Basel (UKBB), University of Basel.
authors.forename: Andrea
authors.initials: A
authors.lastname: Zelmer
authors.affiliation: University Children's Hospital Basel (UKBB), University of Basel; Department of Clinical Research, University of Basel.
authors.forename: Sven
authors.initials: S
authors.lastname: Wellmann
mesh_terms: D000906:Antibodies; D015153:Blotting, Western; D006801:Humans; D047468:Immunoprecipitation; D050779:Nuclear Factor 90 Proteins / Q000737:chemistry; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry; D015398:Signal Transduction
chemical_list: D000906:Antibodies; C429643:ILF3 protein, human; D050779:Nuclear Factor 90 Proteins; D011506:Proteins; C099506:RBM3 protein, human; D016601:RNA-Binding Proteins