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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Traffic. 2014 Oct 27;15(12):1344–1365. doi: 10.1111/tra.12229

Figure 9. Cu washout initiates retrograde traffic (apical to TGN) of ATP7B, which is largely unaffected by Baf.

Figure 9

ATP7B was staged at the apical region of WIF-B cells with Cu (see Figure 7A). Cells were then pre-incubated for 30 minutes in DMSO or Baf to deacidify intracellular compartments [see Figure 7B (+DMSO) and Figure 7E (+Baf)]. Cells were thereafter rinsed and incubated for an additional 180 minutes in 10 μM BCS to initiate retrograde traffic of ATP7B. A-C) Kinetics of ATP7B retrograde trafficking in presence of DMSO or D-F), 50 nM Baf. Cells were fixed at various times after initiating retrograde trafficking then triple-stained with antibodies to ATP7B (green), EEA1 (red) and APN (blue), analyzed and imaged by confocal microscopy. A-F) Single planes from the nuclear region. C' and F') Single planes from the peripheral region. G) Image analysis was used to quantify the fraction of ATP7B that overlapped with EEA1, or H) TGN38. Data for each time point represent 4-7 confocal stacks from 1 or 2 independent experiments, depending on the time point.