Figure 8.

O-Glycosylation of MAL does not interfere with its ability to gain access to GEMs. (A) COS-7 cells transiently expressing MAL-T cells were surface biotinylated 48 h after transfection and lysed with 1% Triton X-100 plus octyl-glucoside at 4°C. The extract was then subjected to immunoprecipitation with mAb 9E10, and the immunoprecipitate was incubated in the absence or presence of neuraminidase. After SDS-PAGE analysis, surface MAL-T was visualized with streptavidin-peroxidase. The positions of the sialylated (S) and asialo (A) MAL-T species are indicated. (B) COS-7 cells transiently expressing MAL-T cells were extracted with 1% Triton X-100 at 4°C at 48 h after transfection and subjected to centrifugation to equilibrium in sucrose density gradients. Aliquots from each fraction were subjected to immunoblot analysis with mAb 9E10. The positions of the sialylated (S) and asialo (A) MAL-T species are indicated. The position of a third band (*), detected by immunoblot analysis but not by surface labeling, likely corresponds to nonglycosylated MAL-T species.