FIGURE 8.

Functionality of LDLR-WT and LDLR-R410S. A–C, HEK293 cells transfected with LDLR-WT (WT) or LDLR-R410S (RS) were evaluated. A, ¹²⁵I-LDL binding to LDLR after a 1-h incubation at 4 °C with increasing concentrations of ¹²⁵I-LDL (200 cpm/pg). B, time-dependent ¹²⁵I-LDL internalization after continuous incubation with 2 ng of ¹²⁵I-LDL at 37 °C. C, time-dependent ¹²⁵I-LDL degradation after continuous incubation with 2 ng of ¹²⁵I-LDL (4 ng/ml) at 37 °C. At each time point, the medium was precipitated with ice-cold TCA, and the TCA-soluble material was used as a measure of the degraded ¹²⁵I-LDL. Data are representative of three independent experiments each performed in four biological replicates. Points are the averages ± S.D. within one experiment. D and E, HepG2 cells overexpressing WT or RS were incubated with DiI-LDL (5 μg/ml) and analyzed by confocal microscopy as follows: DiI-LDL binding to the cell surface after 4-h incubation at 4 °C (D) or DiI-LDL internalization after 4 h at 37 °C (E). Quantifications in D and E were derived from analyses of 12 transfected cells (EGFP-positive)/condition/experiment. Scale bar, 15 μm. Data are representative of at least two independent experiments. Bars are averages ± S.D. **, p < 0.01 (t test).