Figure 5From: Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularlyEfect of hypertonic, conditioned medium on internalization of LDL and on the amount of cell surface LDLR. HepG2 cells were transiently transfected with empty plasmid, WT-PCSK9-His plasmid or D374Y-PCSK9-His plasmid. Conditioned media from transfected cells were made hypertonic by addition of NaCl which increased the final concentration of NaCl by 150 mmol/l. The hypertonic medium was added to untransfected HepG2 cells for a 3 h incubation at 37°C. Isotonic, conditioned medium which had not been added NaCl was used as control. A) To measure internalization of LDL the cells were added fluorescently labelled DiD-LDL (10 μg/ml) and incubated for 2 h at 37°C. B) To measure the amounts of cell surface LDLR, the cells were labelled with anti-LDLR IgG-C7 antibody and counter-stained with Alexa Fluor® 488 goat anti-mouse IgG. Cell fluorescence was quantified by flow cytometry. Results represent mean (± SD) of three experiments (* p < 0.05, ** p < 0.01, *** p < 0.001).Back to article page