FIGURE 4:

Depletion of adaptor protein AP-1 results in BACE1 accumulation in the TGN. (A) HeLa cells stably expressing BACE1-GFP were transfected with either control siRNA, AP-1γ siRNA, GGA1 siRNA, AP-4ε siRNA, or Arl5b siRNA for 72 h. (B) HeLa cells stably expressing Arl5b(Q70L)-GFP were transfected with either control siRNA or Arl5b siRNA for 72 h. Cells in A and B were lysed in RIPA buffer, and 20 μg of cell extracts was subjected to SDS–PAGE on 4–12% gradient polyacrylamide gel. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and probed with (A) mouse anti–AP-1γ, rabbit anti-GGA1, mouse anti–AP-4ε, (B) mouse anti-GFP, and (A and B) mouse anti–a-tubulin antibodies using a chemiluminescence detection system. (C) Monolayers of HeLa cells were fixed, permeabilized, and blocked, followed by staining with mouse anti–golgin-97 antibodies (red) and DAPI (blue). Higher magnification of the merged images of the cells marked * are also shown. Bar represents 10 μm. (D) The percentage of BACE1-GFP at the TGN was calculated as a percentage of total BACE1-GFP pixels that overlapped with golgin-97. Data are represented as the mean ± SD of three independent experiments (n = 15) and analyzed by one-way ANOVA using Tukey’s test. *** p < 0.001.