FIGURE 7.

Effect of loss of Arg⁴¹⁰ on LDLR expression, functionality, and sensitivity to extracellular PCSK9-mediated degradation. A–D, HEK293 cells transfected with LDLR-WT (WT) or LDLR-R410 mutants (RS, RK, RE, RA) were analyzed for total LDLR expression by ELISA (A), cell surface LDLR expression by FACS (B), sensitivity of LDLR to endoglycosidase H, by WB (C), and cell surface LDLR expression, by FACS, following an 18-h incubation with conditioned media from HEK293 cells: no PCSK9 control media (Cnt), or PCSK9 media (∼1.1 μg/ml), PCSK9-WT (PC9-WT), or GOF PCSK9-DY (PC9-DY) (D). E and F, immunofluorescence microscopy in HepG2 cells transfected with WT or RS, RK, RE, RA mutants. E, total LDLR, under permeabilized conditions (PERM), and cell surface LDLR, under non-permeabilized conditions (NON PERM). F, DiI-LDL internalization after 4-h incubation with 5 μg/ml DiI-LDL at 37 °C. Quantifications in E and F were derived from analyses of 12 transfected cells (EGFP-positive)/condition/experiment. Scale bar, 15 μm. Bars in A, B, and F are averages ± S.D. of five independent experiments, and bars in C and D are averages ± S.D. of two independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (t test).