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. 2018 Sep 25;7:e38839. doi: 10.7554/eLife.38839

Figure 5. SURF4 mutagenesis causes a decrease in PCSK9 extracellular secretion and an accumulation of PCSK9 in the ER.

(A) Fluorescence detection of PCSK9-eGFP in extracellular conditioned media relative to cellular lysate in WT (n = 7) and clonal SURF4-deficient (n = 5) fluorescent reporter cell lines. (B) Immunoblotting of tetracycline-inducible PCSK9 in extracellular conditioned media and cellular lysates from WT, SURF4-deficient, or SURF4 rescued cells. (C) Quantification of densitometry of native PCSK9 relative in conditioned media and cellular lysates, normalized to GAPDH, for WT, SURF4-deficient, or SURF4 rescued cells (n = 4 biologic replicates for each cell line). (D) Quantitative PCR of SURF4 and PCSK9 transcript levels from RNA isolated from a SURF4 WT or mutant fluorescent reporter cell line (n = 3 measurement replicates). (E) Live cell fluorescence microscopy of fluorescent reporter cells, either WT or SURF4-deficient, transfected with the ER marker ERoxBFP. Scale bar = 10 µm. (F) Quantification of PCSK9-eGFP signal intensity in pixels positive for ERoxBFP fluorescence (n = 14 for each cell line). (G) EndoH-sensitivity of PCSK9 expressed in WT, SURF4-deficient, or SURF4 rescued cells. (H) Quantification of endoH-sensitivity, normalized to average intensity of average WT band intensity (n = 4 biologic replicates for each cell line). *p<0.05 by Student’s t-test. Error bars represent standard deviations.

Figure 5.

Figure 5—figure supplement 1. Pulse-chase labeling of PCSK9-eGFP secretion.

Figure 5—figure supplement 1.

A SURF4 mutant cell line with a tetracycline-inducible PCSK9-eGFP construct and with (SURF4 rescue) or without (SURF4 mutant) a stably integrated SURF4 cDNA were radiolabeled with tracer containing 35S-Met and 35S-Cys. Conditioned media and cellular lysates were harvested at the indicated time points and PCSK9-eGFP immunoprecipitated and analyzed by SDS-PAGE electrophoresis and autoradiography.
Figure 5—figure supplement 2. EndoH-sensitivity of PCSK9 expressed in WT, SURF4-deficient, or SURF4 rescued cells for each of 4 independent biologic replicates.

Figure 5—figure supplement 2.