Affinity chromatography of PCSK9-His from conditioned medium. Conditioned media from HepG2 cells transiently transfected with empty plasmid, wild-type (WT)-PCSK9-His plasmid or D374Y-PCSK9-His plasmid were loaded on affinity columns containing a nickel-chelating resin. Conditioned medium before and after affinity chromatography was subjected to western blot analysis using an anti-PCSK9 antibody. Western blot analyses of cell lysates equivalent to 20 μg of protein per lane, were included to identify pro-PCSK9 and mature, cleaved PCSK9. Four independent experiments were performed from which one representative western blot is shown.