Fig. 5.

Binding of LDL to the WT and mutant LDLR. A, B: Expression of the WT and mutant LDLR in Hepa1c1c7 cells. Briefly, the same amount of total proteins isolated from Hepa1c1c7 cells transiently expressing the WT or mutant LDLR were subjected to immunoblotting using a monoclonal anti-LDLR Ab, HL-1, and a monoclonal anti-actin Ab. The relative densitometry of LDLR was the ratio of the densitometry of LDLR to that of actin. Values were mean ± SD of three experiments. C, D: LDL uptake. Briefly, Hepa1c1c7 cells transiently expressing the WT or mutant LDLR were incubated with Dil-LDL in the presence or absence of LDL. After washing, the fluorescence signal was measured. The relative fluorescence units (RFU) were normalized to total proteins (micrograms). The amount of specific LDL uptake was the difference between the total counts measured in the absence of unlabeled LDL and the counts measured in the presence of an excess of unlabeled LDL (nonspecific background fluorescence).