Figure 4

Effect of conditioned medium containing PCSK9 on a truncated LDLR (EC-LDLR-His) and on the LDLR in membrane fractions. A) Medium containing a secreted, truncated LDLR (EC-LDLR-His) missing the cytosolic and transmembrane domains of the LDLR was concentrated and incubated for 3 h at 37°C with conditioned media from HepG2 cells transiently transfected with empty plasmid, WT-PCSK9-His plasmid or the D374Y-PCSK9-His plasmid. B) HepG2 cell membrane fractions were isolated by ultracentrifugation and incubated for 3 h at 37°C with conditioned media from HepG2 cells transiently transfected with empty plasmid, WT-PCSK9-His plasmid or the D374Y-PCSK9-His plasmid. After incubations, both EC-LDLR-His and membrane fractions (15 μg per lane) were subjected to western blotting in order to determine the amout of LDLR. For the membrane fractions, the amount of transferrin receptor (TFRC) was used as control. Three independent experiments were preformed from which one representative western blot is shown.