Maxibinding immunoplate

For the Dvl–CXXC5 in vitro binding assay, 100 μl of 5 mg/ml purified Dvl PDZ domain was added into each well of a 96‐well Maxibinding Immunoplate (SPL, Seoul, Korea) and incubated overnight in a 4°C chamber. After washing with phosphate‐buffered saline (PBS), 100 μl of 10 μM PolyR‐DBM (Kim et al, 2015) was added to each well and incubated for 4 h in a 4°C chamber. After washing with PBS 3 times, 100 μl of 2 μM small‐molecule compounds in PBS was added to each well and incubated for 4 h at room temperature. After washing with PBS, the fluorescence of each well was measured using a Fluorstar Optima microplate reader (BGM Lab Technologie, Ortenberg, Germany). Small molecules used in screening were from in‐house collection of Korea Research Institute of Chemical Technology (Daejeon, Korea) or were purchased from ChemDiv Inc. (San Diego, CA).

Virus particles (1 × 10⁵ plaque-forming unit PFU/well) in PBS buffer were coated onto a Maxibinding Immunoplate (SPL Life Sciences, Republic of Korea) and incubated at 4°C overnight. The plate was washed five times with TBS-T and blocked with blocking buffer (5% skim milk in TBS-T buffer) for 2 h at RT. Then, 100 μL of the indicated proteins in a 2X serial dilution (starting from the 100 μg/well) containing blocking buffer was added and incubated at 37°C for 1 h. Polyclonal rabbit anti-HA antibodies (1:3000 dilution; Invitrogen, USA) were used as a control to ensure that the virions were present and intact for the assay. The proteins were detected using anti-6X His tag antibodies at a 1:3000 dilution (Abcam, UK) for 2 h at RT. After washing, 100 μL of a 1:5000 goat anti-mouse IgG H&L (HRP) (Abcam, UK) dilution for anti-6X His tag, and goat anti-rabbit IgG-HRP conjugated antibodies (Invitrogen, USA) for anti-HA antibodies were added and incubated for 2 h at RT. The TMB substrate and 1 M sulfuric acid were added last.

Influenza virions were diluted in PBS and coated onto an maxibinding immunoplate (SPL Life Sciences, Republic of Korea) at 4°C overnight (1 × 10⁵ plaque-forming unit PFU/well). The NVLH8 protein (100 μL/well) in a 2X serial dilution (starting from the 100 μg/well) was added to the plate after incubation with a blocking buffer (5% skim milk in TBS-T buffer) for 2 h at RT. Following an incubation at 37°C for 1 h, 100 μL anti-6X His tag antibodies at a 1:3,000 dilution (Abcam, United Kingdom) were added to the TBS-T-washed plate for 2 h at RT. The virions coated in the plate were validated using polyclonal rabbit anti-HA antibodies (1:3,000 dilution; Invitrogen, United States). Goat anti-mouse IgG H&L (HRP) (1:5,000 dilution, Abcam, United Kingdom) and goat anti-rabbit IgG-HRP-conjugated antibodies (1:5,000 dilution, Invitrogen, United States) were added and incubated for 2 h at RT. After adding TMB and 1 M sulfuric acid, the plate was ready at OD450. The experiment was reproduced three different times independently. Each batch was designed with five data points, and three data points were chosen to be analyzed.