Figure 2
Functional characterization of novel LDLR variants.
(A). LDLR expression. Cells were transfected with the corresponding plasmids carrying the mutations of interest, LDLR was overexpressed for 48 h and then cells were analyzed by Western blot. Whole cell extracts (20 μg) were fractioned in non reducing 8.5% SDS-PAGE, transferred onto nitrocellulose membranes for incubation with a rabbit polyclonal anti-hLDLR antibody and detected by chemioluminiscence as described in Methods section. A representative experiment from three independently performed assays is shown in upper panel. (B–D): Analysis of variant functionality by FACS: LDLR expression at cellular membrane (using WT as example, B); LDL binding after 4 h incubation at 4 °C (using WT as example, C); and LDL internalization efficiency after 4 h incubation at 37 °C (using WT as example, D). (E) Functional characterization of LDLR variants. LDLR expression, LDL binding and LDL uptake. 10,000 cells were acquired in a Facscalibur and values of LDL uptake, binding and LDLR expression were calculated as described in Methods. The values represent the mean of triplicate determinations (n = 3); error bars represent ± SD.
