FIGURE 6:

GGA1 depletion reduces trafficking of BACE1 S498D mutant from the early endosome to the recycling endosome. (A–C) HeLa cells were transfected with either control siRNA or GGA1 siRNA for 72 h and transfected with either wtBACE1 or BACE1 phosphomutants constructs for a further 24 h. Transfected cells were incubated with anti-BACE1 antibodies on ice for 30 min. For the 0-min time point, cells were washed in cold PBS and immediately fixed and permeabilized. Antibody-BACE1 complexes were internalized by shifting the temperature to 37°C for indicated times before fixation and permeabilization. Cells were stained for the internalized BACE1-antibody complexes with Alexa-conjugated secondary antibodies (red) and either EEA1 or Rab11 using mouse monoclonal antibodies to EEA1 or mouse monoclonal antibodies to Rab11 (green). (A) Confocal microscopic images after 60 min at 37°C. Bar represents 10 µm. (B, C) The percentage of wtBACE1 and the BACE1 phosphomutants at the early endosomes and recycling endosomes at each time point was calculated from the percentage of total BACE1 pixels that overlapped with (B) EEA1 or (C) Rab11, respectively. All calculations were performed using the OBCOL plug-in on ImageJ (n = 15 for each marker and timepoint from three independent experiments). Error bars represent SEM. *p < 0.05, ***p < 0.001,