FIG. 3.

US8 partially colocalizes with cellular endosomal-lysosomal proteins. (A) U373+US8-HA cells were pulsed with [³⁵S]methionine-cysteine for 5 min and chased with nonradioactive medium for the time periods indicated. The cells were lysed in 0.5% NP-40 and immunoprecipitated with antiserum specific for the HA epitope. After immunoprecipitation, half of each sample was treated with endo H (+EndoH) while half was left untreated (−EndoH). Apparent positions of molecular mass markers are indicated on the left (in kilodaltons). (B) Fixed, permeabilized U373+US8-HA cells were incubated with either a primary monoclonal antibody specific for the HA epitope (US8-HA); monoclonal antibody W6/32, which recognizes folded MHC class I molecules (Class I); a monoclonal antibody that recognizes the human transferrin receptor (TFN-R); or a monoclonal antibody specific for the human ER chaperone, calnexin. The cells were then incubated with an Alexa-568-conjugated secondary antibody (red fluorescence) and the DNA-binding dye DAPI (4′,6′-diamidino-2-phenylindole; blue fluorescence) (Sigma) prior to analysis with a Bio-Rad MRC1024 laser-scanning confocal microscope. (C) Fixed, permeabilized U373+US8-HA cells were incubated with either a monoclonal antibody that recognizes the HA epitope, a monoclonal antibody specific for the lysosomal protein Lamp-1, or a rabbit polyclonal antiserum generated against the luminal domain of US8. The cells were then incubated with either an Alexa-568-conjugated anti-mouse secondary antibody (red fluorescence) or an Alexa-488-conjugated anti-rabbit secondary antibody (green fluorescence). Some cells were incubated with fluorescently labeled human transferrin at 37°C prior to fixation of the cells (TFN-R; green fluorescence). Images were obtained with a Spot RT digital camera mounted on a TE300 Nikon microscope. Merged two-color overlays demonstrating the colocalization of internalized transferrin, Lamp-1, and US8 are shown.