Pooled human umbilical vein endothelial cells (HUVECs) were cultured in EGM-2 (endothelial growth medium, consisting of EBM-2 (endothelial basal medium) supplemented with the EGM-2 bullit kit (Lonza). Cells were maintained under standard growth conditions (at 37°C, 5% CO2) and used for experiments between passage 3-5. All experiments were preceded by a 2-hour serum starvation in EBM-2 supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin (pen/strep). Treatment of cells was performed in EBM-2 supplemented with 0.2% fetal calf serum (FCS) and pen/strep (Invitrogen). To determine the condition of the cells, a viability assay was performed using Cell Proliferation reagent WST-1 (Roche).

For biosynthesis inhibition, 18 µM cycloheximide (Sigma-Aldrich) were pre-incubated with cells for 2 hours before adding GrK or GrKSA. All were directly added to the wells for a 24 hour-treatment. Recombinant human VEGF165 protein was from R&D Systems. ATAP2 was from Santa Cruz Biotechnology.

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