FIG. 4.

Us9-EGFP is maintained in the TGN region by retrieval of molecules from the plasma membrane. Glass coverslips plated with PK15 cells stably expressing Us9-EGFP were cooled and incubated with GFP-specific antiserum on ice for 30 min. The cells were then shifted to 32°C by the addition of prewarmed medium and placement in a 32°C incubator. At 0 min (A), 15 min (B), 30 min (C), 60 min (D), or 90 min (E), the cells were fixed with paraformaldehyde and the localization of the GFP antiserum was detected with an Alexa 568-conjugated goat anti-rabbit IgG secondary antibody (red). The red fluorescence represents the localization of internalized Us9-EGFP molecules, and the green fluorescence represents the total pool of Us9-EGFP molecules. Panel F shows the localization of internalized Us9-EGFP after 90 min of temperature shift to 37°C. All images are confocal sections taken through the center of the cell.