Figure 3.

(A) Immunofluorescence on mottled fibroblast clones stably transfected with Wilson cDNA mutants H1069Q and H1069A without copper treatment. Cells were processed for indirect immunofluorescence by using antibodies against the WD protein or protein disulfide isomerase. (×600.) (B) Pulse–chase labeling of Wilson protein endogenously expressed in HepG2 cells or stably expressed in mottled fibroblast clones as the wild-type protein or mutants H1069Q and H1069A. Cultures were pulsed for 30 min with [³⁵S]methionine and [³⁵S]cysteine and then chased for the indicated times in media containing excess methionine and cysteine. Immunoprecipitates of cell lysates were analyzed by fluorography after SDS/7.5% PAGE and were quantified by using a Phosphorimager (Molecular Dynamics).