Fig. 4. F_c1-37 recycles from the plasma membrane to a membrane compartment that overlaps the distribution of the mannose 6-P receptor. MDCK cells stably expressing F_c1-37 were incubated with the anti-F_c receptor antibody for 1 hour at 4°C. Following washing with cold DMEM, the cells were incubated for 15 minutes (A,C,E) or 45 minutes (B,D,F) at 37°C. At each time point, the cells were fixed, permeabilized and incubated with a mouse monoclonal directed against the mannose 6-phosphate receptor (M-6-P). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine and goat anti-mouse IgG conjugated to FITC. Following washing, the localization of F_c1-37 (A,B) and the mannose 6-phosphate receptor (C,D) was visualized on a Zeiss LSM 510 laser-scanning microscope. The merged images showing the overlap of F_c1-37 and the mannose 6-phosphate receptor are shown in E and F. The arrows in A indicate the F_c1-37 chimeras that still reside on the cell surface following a 15 minute incubation at 37°C. Bar (A,B), 10 µm.

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