Figure 1.

Folded K^d molecules were co-localized with APLP2 in Rab5⁺ early endosomes. (A) APLP2 and endocytosed K^d were co-localized with endosomal marker Rab5. (B) APLP2 and endocytosed K^d were co-localized with the Rab5 dominant negative mutant Rab5Q79L-GFP, which causes early endosomes to be enlarged. HeLa cells stably transfected with K^d and transfected with either Rab5 or Rab5Q79L-GFP were pulsed with anti-K^d Ab 34-1-2 and warmed for 15 min at 37°C. The cells were then incubated with 0.5% acetic acid/500 mM NaCl to strip non-internalized surface-bound 34-1-2 Ab. The cells were fixed with 4% paraformaldehyde, and incubated with rabbit anti-APLP2 serum in staining solution containing saponin, washed, and incubated with fluorescently labeled secondary antibodies in the same staining solution. The images were analyzed on a Zeiss LSM 5 Pascal confocal microscope. Red = APLP2; green = Rab5 or Rab5Q79L; blue = K^d; white = co-localized APLP2, K^d, and Rab5 or Rab5Q79L. Bar indicates 10 μm. Insets display more highly magnified images of the areas shown in the larger boxes, and the arrows in A indicate vesicles in which APLP2, Rab5, and K^d are co-localized.