Human neuropilin 1 fc

Plates (96 well, Nunc Maxisorp; Thermo Fisher Scientific, Waltham, MA, USA) were coated with human Neuropilin-1-Fc (10 ng per well, Cat# 50-101-8343, Fisher, Hampton, NH) and incubated at room temperature overnight. The following day, the plates were washed and blocked with 3% BSA in PBS to minimize non specific adsorptive binding to the plates. SARS-CoV2 Spike protein (100 nM, S1 domain aa16–685, Cat# Z03485, Genscript, Piscataway, NJ), EG00229 (Cat#6986, Tocris) or the indicated compounds were added at 12.5μM and incubated for 30 min at room temperature prior to adding biotinylated human VEGF-A165 (Cat#BT293, R&D systems) at 10 nM. As a negative control, some wells received PBS containing 3% BSA. The plates were incubated at room temperature with shaking for 1 h. Next, the plates were washed with PBS to eliminate unbound protein. Bound biotinylated VEGF was detected by streptavidin-HRP (Cat#016-030-084, Jackson immunoResearch). Tetramethylbenzidine (Cat#DY999, R&D Systems, St. Louis, MO) was used as the colorimetric substrate. The optical density of each well was determined immediately, using a microplate reader (Multiskan Ascent; Thermo Fisher Scientific) set to 450 nm with a correction wavelength of 570 nm. Data were normalized to the background and to the signal detected for VEGF-A165 alone.