Figure 1.

Study of B-fragment colocalization with established markers of the endocytic pathway during incubation at 19.5°C. The following proteins were incubated with HeLa cells at 19.5°C for 1 h: (A) Tf (green) and B-fragment (red), a large arrow indicates a region of perinuclear staining; (B) EGF (red) and B-fragment (green), arrows point out regions where B-fragment and EGF staining are juxtaposed; (D) TF (green) and EGF (red); (E) Dex3 (green) and B-fragment (red), note vesicular (large arrows) and tail-like (small arrows) Dex3 staining. For marker concentrations see Materials and Methods. Digital images (four integration frames) were acquired by confocal microscopy. The right panel represents the superposition of the red and green images. Insets show selected areas at higher magnification. In C, EGF (red) and B-fragment (green) were internalized at 19.5°C, as in B. The cells were then shifted to 37°C for 10 min before fixation. Note that B-fragment and EGF-specific labeling did basically not overlap.