Gel filtration of conditioned medium from HepG2 cells transiently transfected with D374Y- PCSK9 -FLAG plasmid. Conditioned medium from HepG2 cells transiently transfected with D374Y-PCSK9-FLAG plasmid or with empty plasmid were concentrated approximately 10 times and 1 ml was loaded onto a Superdex 200 gel filtration column. Fractions of 2 ml were collected. HepG2 cells were incubated with the different fractions to determine the effect on internalization of LDL by flow cytometry. Fractions containing molecular weight standards are indicated on the x-axis. Aliquotes of fractions 15–21 were also subjected to precipitation of proteins by trichloroacetic acid. The amount of mature D374Y-PCSK9-FLAG in the fractions was determined by western blot analysis using an anti-FLAG antibody as shown in the insert.