The direct observation of Upf2-EJC complex formation was performed using a Duolink® kit (Olink Bioscience, Uppsala, Sweden; product now owned by Sigma-Aldrich) following the manufacturer's protocol (23 (link)). This method enables the visualization of complex formation in cells by proximity ligation of single-stranded DNA conjugated with a secondary antibody (24 (link)), and is available for the complex in nuclei and cytoplasm (25 (link)). Signal can be observed when the distance between the secondary antibodies is <40 nm. Therefore, this method can detect not only direct protein-protein interactions, but also complex formation, assuming that the bound first antibodies are proximal enough. The first antibodies used in the present study were as described in the above Immunostaining and observation section. Images were captured with either an Axiovert 200 M inverted fluorescence microscope or an LSM 710 confocal point-scanning microscope (Carl Zeiss, München, Germany).