Antibody labeling and binding assay with b1b2 domain of NRP-1

Anti-NRP1 monoclonal antibodies were labeled with DyLight™ 488 NHS Ester (DL488; #46402, Thermo Fisher Scientific). First, the mAbs were purified of glycerol by dialysis in 3L of 1X PBS for 2 days at 4 °C, using 3.5K MWCO Slide-A-Lyzer™ Dialysis Cassettes (#66330, Thermo Fisher Scientific). Next, 500 μl of 0.6 mg/ml mAb1, mAb2 or mAb3 was incubated with 7 μl of 2 mM dye in DMF for 2 h at RT. Excess of dye was removed using 3K MWCO Protein Concentrators PES (#88512, Thermo Fisher Scientific) by centrifugation at 14,000 × g at RT for 30 min and topping off with PBS; the washes were repeated 3 times. For cell-free binding studies, hexahistidine-tagged (6xHis-tagged) recombinant b1b2 domain of NRP-1, or the mutant NRP-1 b1b2 with an inactive CendR-binding pocket, (0.5 mg/ml; in-house) was coated onto Ni-NTA magnetic agarose beads (5% solution; #36113, Qiagen GmbH) by incubating with end-over-end mixing at RT for 1 h. PBS containing 0.05% of Triton X-100 (Sigma Aldrich) and 0.2% BSA (GE Healthcare) was used as the washing buffer. Protein-coated magnetic beads were incubated with DL488-labeled mAb1, mAb2 or mAb3 (40 μg/ml) with end-over-end

mixing at RT for 1 h, followed by 3 washes with the washing buffer, and the release of protein-mAb complexes with imidazole elution buffer [washing buffer + 400 mM imidazole (Sigma Aldrich)]. A Victor X5 Multilabel Plate Reader (PerkinElmer) with a 488/515 nm filter set was used for the fluorescence measurements, and all measurements were done in triplicates.