Figure 6.

Mutation of the HIC⁸¹⁸ sequence reduces the rate of β-VLDL uptake by the Y807C LDLR by half—LDLR^−/− fibroblasts were infected with bicistronic retroviruses encoding just GFP (vector) or the normal, Y807C, HIC⁸¹⁸>AAA or Y807C+HIC⁸¹⁸>AAA variants of the LDLR, together with GFP. Cells were sorted based upon green fluorescence to >95% homogeneity. (A) A 5 μg weight of total cell lysate from each fibroblast was run on SDS–PAGE gels, transferred to nylon membranes and immunoblotted for the LDLR or for CD44. (B, C) Cells were incubated with 10 μg/ml ¹²⁵I-β-VLDL for 1 h at 4°C, and then shifted to 37°C for 0, 5, 10 or 15 min. Internalized and surface-bound ¹²⁵I-β-VLDL were separated as described in Materials and methods, and the ratio is presented in panel B Individual values for internalized and surface-bound β-VLDL are presented in panel C Each point is the mean of four determinations. Error bars show the standard deviation. The experiment shown is representative of three independent experiments.