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Figure 3.
EGF-AB(H306Y) blocks uptake of PCSK9 in HuH7 cells. HuH7
cells were treated 30 min with 5 μg/ml purified full-length
FLAG-tagged PCSK9 alone or in combination with 5 μm
EGF-AB(H306Y) blocking peptide or EGF-AB(L318A) negative-control
peptide. A–D, cells were immunostained for PCSK9 (green) and
nuclear stained with 4′,6-diamidino-2-phenylindole (magenta)
as described under “Experimental Procedures.” E, cells were
lysed and subjected to SDS-PAGE and immunoblotting for
FLAG-tagged PCSK9 as described in the legend to Fig. 1B. Actin
was detected as a control for equal protein loading. PCSK9 and
actin levels were visualized and quantified using an
IRDye800-labeled secondary antibody and the LI-COR Odyssey
infrared imaging system. PCSK9 levels were normalized to actin
expression.
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