Fig. 4: Determination of biligand cellular uptake mechanisms.
A NIH-OVCAR-3 cells were treated with 200 nM fluorescein-labeled B7,3,5 and B5,3,5 at 37 °C. B Cells were treated with biligands at 4 °C to inhibit energy-dependent processes; C Cells were pre-treated with 2-deoxy-D-glucose to inhibit ATP uptake. D–G. NIH OVCAR3 cells were pre-treated with specific endocytosis inhibitors for 30 min. The inhibitors used were: 10 μM cytochalasin D (macropinocytosis, D), 4 μM filipin (caveolae-dependent endocytosis, E), chlorpromazine (clathrin-dependent endocytosis, F) or 5 mM methyl-β-cyclodextrin (lipid raft-mediated endocytosis, G). Cells were then treated for 8 h with 200 nM fluorecein-labeled B7,3,5 and B5,3,5. Following the incubation, cells were washed, trypsinized, and sorted using flow cytometry.
