Visualizing Protein-Protein Interactions

The VIP assay and subsequent immunoblotting analysis were performed by a previously described method (Katoh et al., 2015 (link), 2016 (link)) with minor modifications (Nishijima et al., 2017 (link)). Briefly, approximately 1.6×10⁶ HEK293T cells in six-well plates were transfected with EGFP and mChe fusion constructs using Polyethylenimine Max (20 µg, Polysciences, Warrington, USA), and cultured for 24 h. The transfected cells were then lysed for 20 min on ice in 250 µl of HMDEKN cell lysis buffer [10 mM HEPES (pH 7.4), 5 mM MgSO₄, 1 mM DTT, 0.5 mM EDTA, 25 mM KCl, and 0.05% NP-40] containing EDTA-free protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan), and centrifuged at 16,100×g for 15 min at 4°C in a microcentrifuge. The supernatants (200 µl) were incubated with 5 µl of GST–anti-GFP Nb beads for 1 h at 4°C. After washing three times with 180 µl of the cell lysis buffer, the precipitated beads were observed using an all-in-one-type fluorescence microscope (BZ-8000, Keyence, Osaka, Japan) using a 20×/0.75 objective lens under constant conditions (sensitivity ISO 400, exposure 1/30 s for green fluorescence; and sensitivity ISO 800, exposure 1/10 s for red fluorescence).