Proximity Ligation Assay for Protein Interactions

The proximity ligation assay (PLA), which enables detection of protein interactions within a cell, was performed according to the Duolink kit method (Nacalai Inc., Kyoto, Japan) as described previously (Okazaki et al., 2020 (link)). We applied the PLA method to examine a close association between Cp110 and GFP–Orbit. For detection of the complexes, we used anti-Cp110 antibody (1:300; a gift from Jordan Raff) and anti-GFP antibody (1:200; A-6455; Thermo Fisher Scientific, Waltham, USA) to detect complexes containing Cp110 and Orbit. Samples were observed with a fluorescence microscope (IX81; Olympus, Tokyo, Japan). Image acquisition was controlled through the Metamorph software version 7.6 (Molecular Devices) and processed with ImageJ version 1.51 or Adobe Photoshop CS4.