Viaflo assist

An ELISA-based assay was used to identify novel ligands for Unc5D-AP. The experiment was done in duplicate. Twenty μL of a solution at 3 μg/mL of mouse anti-AP in 1X PBS was added to each well of 96-well plates using an automated multichannel pipette (Viaflo Assist, Integra), sealed and incubated overnight at 4C. The following day, the plate was washed once with PBS and 1% casein was added as a blocking agent, which was removed after 1 h at room temperature using an automated microplate washer (HydroSpeed, Tecan). Next, to each well, 20 μL of Unc5D-AP conditioned medium, containing 2 μL of monoclonal mouse anti-human IgG1-HRP was added using an automated plate copier (Viaflo96, Integra) along with 20 μL of culture medium from 95 different ecto-Fc prey proteins. Plates were sealed and incubated for 4 h at room temperature in the dark. Plates were subsequently washed, and 35 μL 1-Step Ultra TMB-ELISA HRP substrate was added using an automated multichannel pipette (Viaflo Assist, Integra); after 1 h incubation at room temperature, the absorbance at 650 nm was recorded with a microplate Spectramax i3 plate reader (Molecular Devices). Finally, plates were scanned to obtain matching images of the 650 nm reading. A positive control (known interactors NRXN/NLGN1) was used (Ozgul et al., 2019 (link)).

The in vitro kinase inhibitory assay was performed at Reaction Biology Corp (http://www.reactionbiology.com accessed on 15 February 2022) using Kinase HotSpotSM in order to measure the inhibitory activities of tested candidates, as previously performed [20 (link),21 (link)]. The experiment contained specific kinase/substrate pairs along with required cofactors. Base reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl₂, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na₃VO₄, 2 mM DTT, 1% DMSO. The tested compounds were dissolved in 100% DMSO to a specific concentration. The serial dilution was conducted by Integra Viaflo Assist in DMSO. The reaction mixture containing the examined compound and 33P-ATP was incubated at rt for 2 h, and radioactivity was detected by the filter-binding method. Kinase activity data were expressed as the percentage of remaining kinase activity in test samples compared to vehicle (DMSO) reactions.

Assays were performed in base reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO). YKL-05–099 was dissolved in 100% DMSO in a 10 mM stock. Serial dilution was conducted by Integra Viaflo Assist in DMSO. Recombinant CSF1R (Invitrogen, Carlsbad, CA) was used at a concentration of 2.5 nM. The substrate used was pEY (Sigma-Aldrich, St. Louis, MO) at a concentration of 0.2 mg/ml. Kinase assays were supplemented with 2 mM Mn²⁺, and 1 µM ATP was added. Assays were performed for 20 min at room temperature, after which time ³³P-ATP (10 µCi/µl) was added followed by incubation for another 120 min at room temperature. Thereafter, radioactivity incorporated into the pEY peptide substrate was detected by filter-binding method. Kinase activity data were expressed as the percent remaining kinase activity in test samples compared to DMSO reactions. IC₅₀ values and curve fits were obtained using Prism 8.0 GraphPad Software (GraphPad Software, San Diego, CA).