Figure 1.

(A) Immunoblot of lysates from mottled fibroblasts (802–1) stably transfected with the wild-type Wilson cDNA. Protein (125 μg) from the parental cell line (lane 1) or two independent clones (lanes 2 and 3) with HepG2 lysate as a positive control (lane 4) were separated by SDS/7.5% PAGE, transferred to nitrocellulose, incubated with Wilson antiserum, and developed with chemiluminescence. (B) Indirect immunofluorescence localization of the wild-type Wilson protein (WD) and ARF with or without copper treatment in the stably transfected mottled fibroblast clone 4, with the parental 802–1 cell line as a negative control. (×600) (C) ⁶⁴Cu retention by mottled fibroblasts (802–1 Mo −/Y), wild-type fibroblasts (802–5 Mo +/Y), and two independent clones of mottled fibroblasts stably transfected with the Wilson cDNA (802–1 WD 4 and 802–1 WD 13). Cells were incubated with ⁶⁴Cu for 72 hr and processed as described in Materials and Methods. (D) Copper toxicity profile of mottled fibroblasts (802–1), wild-type fibroblasts (802–5), and mottled fibroblast clone 4 stably transfected with the Wilson cDNA (802–1 WD). Cells were grown in media supplemented with increasing concentrations of copper and assessed for viability at 72 hr by using an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide-based assay as described in Materials and Methods.