Fig. 6. Endocytosed bPGI/AMF can undergo more than one cycle of endocytosis. NIH-3T3 cells were incubated with 25 µg ml^–1 bPGI/AMF in complete medium (A-F) or with complete medium in the absence of bPGI/AMF (G-I) for 30 minutes at 37°C. The cells were then washed three times with complete medium and then incubated 30 minutes with Texas Red-streptavidin (SA-TR) at 37°C (A-C, G-I) or at 4°C (D-F). Cells were fixed with 3% paraformaldehyde, permeabilized with 0.1% Triton X-100, and labeled with FITC-streptavidin (SA-FITC) to detect endocytosed bPGI/AMF that had not been bound by Texas Red-streptavidin (B,E,H). Texas Red (red) and FITC-streptavidin (green) confocal images were merged and confocal co-localization appears in yellow (C,F,I). When added at 37°C (A-C) but not at 4°C (D-F), Texas Red-streptavidin can be seen to label PGI/AMF positive MVBs and was therefore captured for a second round of endocytosis by recycling bPGI/AMF. Bar, 10 µm.