Effect of nocodazole and ammonium chloride on PCSK9-mediated degradation of the LDLR. HepG2 cells were cultured in media supplemented with nocodazole (20 μg/ml) or ammonium chloride (NH4Cl, 10 mM) for 30 min. The media were then replaced with conditioned media from HepG2 cells transiently transfected with D374Y-PCSK9-FLAG plasmid or with empty plasmid, already containing ammonium chloride or nocodazole, and the incubation was continued for 3 h. The conditioned media had also been added ammonium chloride or nocodazole. Cell membranes were isolated and membrane proteins equivalent to 10 μg were subjected to western blot analysis to determine the amount of LDLR. The amount of transferrin receptor (TFRC) was used as a control. Band intensities were quantified using a ChemiDoc XRS and Quantity One version 4.4.0 software. Results represent the mean (± SD) of four experiments, from which one representative western blot is shown.