FIG. 5.

Us9-EGFP internalization is not an antibody dependent process. PK15 cells stably expressing a Us9-EGFP fusion protein were incubated for 15 min (15′) on ice with Sulfo-NHS-SS-biotin. The cells were then lysed immediately to show the total amount biotinylated (lane T) or shifted to 37°C to allow internalization by the addition of prewarmed medium and placement in a 37°C incubator. At 0 min, 15 min, 30 min, 45 min, or 60 min after the temperature shift, biotin was cleaved from the cell surface with glutathione, cell lysates were prepared, and biotinylated proteins were affinity purified with streptavidin-agarose. The affinity-purified molecules were separated on an SDS–12.5% polyacrylamide gel, transferred to nitrocellulose, and Western blotted with monoclonal GFP antiserum. The migration of molecular mass markers is indicated on the left in kilodaltons.