FIGURE 11.

Pure PCSK9 can generate PCSK9-ΔNx at pH 5.5 with increased activity. A, Coomassie staining of pure PCSK9 purified from the media of baculovirus High Five cells, attesting for its complex formation with the prosegment and its purity as confirmed by protein sequence analysis (data not shown). B, Western blot analysis using our PCSK9 polyclonal antibody (10) of pure PCSK9 (10 μg/ml) incubated in vitro at 37 °C at various pH values for 1 h. Notice the production of PCSK9-ΔNx only at pH 5.5. Following neutralization and incubation with HuH7 cells for 4 h, the cells were lysed, and their content was analyzed by Western blot. C, FACS analysis of cell surface LDLR of HuH7 cells exposed to 10 μg/ml of pure PCSK9 preincubated in vitro at 37 °C at various pH values for 1 h, neutralized, and then incubated with the cells for 4 h.