Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and inlivers of parabiotic mice
J. Clin. Invest. Thomas A. Lagace, et al. 116:2995 doi:10.1172/JCI29383 [
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Figure 4LDLR-dependent endocytosis of PCSK9 in MEFs. (
A) Immunoblot analysis of PCSK9 association with MEFs. Immortalized MEFs derived from wild-type,
Ldlr–/–,
Lrp–/–, and
Ldlr–/–Lrp–/– mice were cultured for 18 hours in medium F prior to treatment with purified human PCSK9 for 4 hours. Cell lysates (30 μg) were subjected to SDS-PAGE and immunoblot analysis using IgG-15A6 to detect PCSK9 or a polyclonal antiserum (Ab 3143) that recognizes the LDLR, as described in Methods. Immunoblots of receptor-associated protein (RAP) were used as a loading control. (
B) Indirect immunofluorescence localization of PCSK9 and the LDLR in wild-type MEFs. MEFs were incubated for 4 hours with 5 μg/ml purified human PCSK9 and processed for double immunofluorescence of PCSK9 (green) and the LDLR (red) as described in Methods. (
C) Indirect immunofluorescence localization of PCSK9, LDLR, and the late-endosomal marker CI-MPR in MEFs cultured in the presence of chloroquine. Wild-type MEFs were incubated for 4 hours with 5 μg/ml purified human PCSK9 in the presence of 0.1 mM chloroquine and processed for double immunofluorescence of PCSK9 (green) and the LDLR (red) or CI-MPR (red) as described in Methods. The merged image shows areas of colocalization of PCSK9 with the LDLR and CI-MPR. Magnification, ×630. Similar results were obtained in 3 independent experiments.
