Figure 7. Removal of Baf partially restores delivery of ATP7B to the apical region in Cu-treated WIF-B cells.

A-B) WIF-B cells treated 60 minutes with Cu plus DMSO or C-D) Cu plus 50 nM Baf. E-F) A parallel set of Baf-treated cells was rinsed and incubated for an additional 180 minutes in 10 μM CuCl₂ plus DMSO before fixation and double-stained with antibodies to ATP7B (green) and APN (blue). Cells were analyzed and imaged by confocal microscopy. A-F) Single planes from the nuclear region. B', D', F') Single planes from peripheral region. G) The extent of overlap between ATP7B and APN at the apical region indicates a partial restoration of apical targeting. H) Washout of Baf restores the number of ATP7B small vesicles to control levels. I) Axial-plane analysis of ATP7B distribution indicates a restoration of the peak ATP7B fluorescence to the typical apical region distribution. Measurements shown in graphs were performed on a minimum of 3 confocal stacks per condition, from a single experiment (approximately 84 cells). J) Western blots of ATP7B from duplicate coverslips in 4 separate experiments indicate no changes in ATP7B protein levels due to new synthesis or aberrant lysosomal targeting due to Baf during the course of the experiment.