Figure 8. Cu-dependent localization of ATP7B at the apical domain is maintained by local recycling through apical endosomes and is perturbed by Baf.

A) WIF-B cells were incubated overnight in 10 μM BCS then switched to 10 μM CuCl₂ for 60 minutes to stage ATP7B at the apical region, then fixed. B-G) Parallel sets of cells were maintained in Cu for an additional 30 minutes, or I-L) 180 minutes in the presence of B-D, I-J) DMSO, or E-G, K-L) 50 nM Baf. Cells were stained with antibodies to ATP7B (green), EEA1 (red) and APN (blue), and imaged by confocal microscopy. A-G and I-L) Single planes from the nuclear region. D', G', J' and L') Single planes from the peripheral region. H) Quantification of the extent of overlap between ATP7B and EEA1 or APN after 30 minutes or, M) 180 minutes in Cu ± Baf. Data shown in H are the mean of 7 confocal stacks from 2 independent experiments (∼196 cells); Data in M represent 3 stacks from a single experiment (∼84 cells). N) Western blots of ATP7B from duplicate coverslips treated as in A, I and K (in 3 separate experiments) indicate no significant changes in ATP7B protein levels.