Biotinylation assay.

HRMECs were transfected with ARF6, ARNO, GEP100, or control siRNA and grown to 100% confluence on 60-mm dishes. Cells were washed twice with room-temperature PBS and incubated with 0.6 mM primaquine (MP Biomedicals) at 37°C for 10 minutes to block recycling during the internalization step (32, 33). The cells were washed twice with ice-cold PBS and labeled with EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) at 0.5 mg/ml in PBS at 4°C for 60 minutes. The excess biotin was removed by washing the cells with ice-cold glycine in PBS with Ca²⁺ and Mg²⁺. The cells were then incubated with 20 ng/ml VEGF and 0.6 mM primaquine at room temperature for 5 minutes. They were then exposed twice to GSH buffer (50 mM glutathione, 75 mM NaOH, 75 mM NaCl, 1 mM EDTA, 0.1% BSA, pH 9.0) on ice for 20 minutes to remove surface biotin. GSH was quenched by washing with 5 mg/ml ice-cold iodoacetamide in PBS with Ca²⁺ and Mg²⁺. After an additional wash with ice-cold PBS, cells were lysed on ice in Pierce IP Lysis Buffer (Thermo Fisher Scientific) with a protease inhibitor cocktail. Cell lysates were centrifuged at 13,000 ×g for 5 minutes, and the supernatant was incubated with High Capacity Streptavidin Agarose Resin (Thermo Fisher Scientific) at 4°C for 60 minutes. Beads were washed 3 times with Pierce IP Lysis Buffer. Bound proteins were released in 2× Laemmli buffer with 5% β-mercaptoethanol at 95°C for 5 minutes, and internalized VEGFR2 was analyzed by immunoblotting with VEGFR2 antibody.