Human scg3

Human Scg3 (Sino Biological) was labeled with biotin using EZ-Link™ Sulfo-NHS-Biotin labeling reagent (NHS-Biotin, Thermo Fisher) according to the manufacturer’s protocol. Briefly, Scg3 in PBS was incubated with NHS-Biotin for 30 min at room temperature, followed by incubation with 1/10 volume of 100 mM Tris-HCl pH 8.0 to stop the reaction. Scg3-biotin was purified using desalt spin columns (Bio-Rad).
Epitope binning was performed to analyze epitope binding specificity of mAbs [18 (link), 19 (link)]. In brief, Scg3-biotin was incubated with streptavidin biosensors (Pall ForteBio). After washing, anti-Scg3 mAbs were incubated with Scg3 on biosensors as blocking mAbs, followed by washing and incubation with different anti-Scg3 mAbs using Octet RED96 instrument (Pall ForteBio) to monitor binding kinetics.

Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza and used for experiments at passage 4–8. Complete classic medium kit with culture boost and attachment factor (CSC, #4 Z0-500) was obtained from Cell Systems. EBM-2 medium (#CC3156) was from Lonza. Pen/Strep (100X) was from Thermo Fisher. Fetal bovine serum (FBS) and trypsin/EDTA were from HyClone. Recombinant human VEGF-165 (#293-VE-010/CF) was from R&D Systems. Human Scg3 (#16012-H08H) was from Sino Biological. PD98059 (#HY-12028) and SCH772984 (#HY-50846) were from MedChem Express.

Antigen affinity-purified anti-Scg3 polyclonal antibody (pAb) was purchased from Proteintech (Rosement, IL). Anti-Scg3 ML49.3 mAb was purified from serum-free conditioned medium of ML49.3 hybridoma as described (LeBlanc et al., 2017 (link)). All antibodies were washed three times with phosphate-buffered saline (PBS) in Amicon centrifugal filter spin units (10 kDa cutoff, Millipore, Billerica, MA). Human retinal microvascular endothelial cells (HRMVECs) and complete classic medium kit with serum and CultureBoost were obtained from Cell Systems (Kirkland, WA) (LeBlanc et al., 2017 (link); LeBlanc et al., 2016 (link)). Human Scg3 were from Sino Biological (Beijing, China). Aflibercept is a drug from Regeneron Pharmaceuticals (Tarrytown, NY).