Fig 2: Expression of PCSK9 variants into culture medium of stably transfected HEK293 cells. PCSK9 secretion was analysed by Western blot. The different PCSK9 variants were purified from the culture medium as described in Materials and Methods and 5 µg total protein was subjected to Western blot analysis. A representative experiment from three independently performed assays is shown. The final line of the blot has been cropped because it is a variant not described in this work. The image is a merge of the nitrocellulose membrane showing the pre-stained MW markers and the signal of the western blot. Image bright has been modified in the whole blot. Original membrane and blot can be found in Supplementary Figures S1 and S2 respectively.

Fig 3: Effect of PCSK9 variants on LDL uptake in (A) transiently transfected HEK293 cells, (B) in HEK293 cells and (C) HepG2 cells incubated with purified PCSK9 variants. LDL uptake in transiently transfected HEK293 cells (A) was determined as described in Materials and Methods. For LDL uptake assay with purified PCSK9 variants, cells were incubated with the PCSK9 variants at 0.5 μg/mL during 2 h prior FITC-LDL addition (B and C). LDL internalization was determined after 4 h incubation at 37 °C as described in Materials and Methods. Values represent the mean ± standard deviation of 3 independent experiments performed by triplicate. *p < 0.01 versus no PCSK9 addition; #p < 0.01 versus wild-type (wt) PCSK9. p.(Asp374Tyr) GOF mutant was used as internal control.