Fig. 5.
Endosomal IDE content of COS-1 cells. (A) Western blot of the PNS and endosomal fractions isolated from COS-1 cells transfected with IDEwt. (B) Western blot of fractions isolated from COS-1 cells transfected with polyanion-binding site mutant IDEK898A,K899A,S901A. (C) Western blot of fractions isolated from COS-1 cells transfected with the distal site mutant IDEY609F. The total protein loaded in each lane is indicated. (D) Green channel indicating labeled endosomes for a representative micrograph (single plane from a 3D image stack), supporting the localization of IDE to endosomes. Endosomes in COS-1 cells were labeled by uptake of Alexa Fluor 488-dextran. A number of cells are visible in the image. (Scale bar: 5 μm.) (E) Red channel indicating IDE distribution for the same micrograph. Expressed IDEwt was labeled by immunostaining with Alexa Fluor 549-conjugated antibody. (F) Merged red and green channels from the same micrograph. In D–F, the Insets show magnified views of the areas indicated by the smaller rectangles to emphasize the degree of dye overlap. (G) Scatterplot showing distribution of red and green channel intensities in voxels from a single COS-1 cell masked in a 3D image stack. The distribution shows a strong spatial correlation between channel intensities consistent with localization of IDE with endosomes. Vertical and horizontal lines indicate threshold cutoffs for statistical analysis. Symbol colors represent the number of voxels in color intensity bins, progressing from blue (lowest number) to red (highest number).