FIGURE 3:

BACE1 phosphomutants in HeLa cells exhibit different rates of transport from the early endosomes. (A, B) HeLa were transfected with either wtBACE1 or BACE1 phosphomutants for 24 h. Transfected cells were incubated with anti-BACE1 antibodies on ice for 30 min, washed in cold PBS, and, for the 0-min time point, immediately fixed and permeabilized. For internalization of the antibody-BACE1 complexes, monolayers were shifted to 37°C for various times before fixation and permeabilization. Cells were then stained for BACE1–antibody complexes with Alexa-conjugated secondary antibodies and either (A) EEA1 or (B) Rab11 using monoclonal antibodies to EEA1 and mouse monoclonal antibodies to Rab11, respectively. The percentage of the BACE1 phosphomutants at the early endosomes or recycling endosomes at each time point was calculated from the percentage of total BACE1 pixels that overlapped with EEA1 or Rab11, respectively. All calculations were performed using the OBCOL plug-in on ImageJ. Data were pooled from three independent experiments and are expressed as the mean ± SEM (n = 15) and analyzed by an unpaired, two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.