Figure 9.

Recycling of MAL from the plasma membrane to the TGN. (A) COS-7 cells were transfected with MAL-T and, 48 h later, surface labeled with sulfo-NHS-biotin for 30 min at 4°C and incubated for 30 min at 4°C in the absence (lane 1) or presence (lane 2) of neuraminidase, or for 15 min at 37°C to allow MAL internalization and then for 1 h with neuraminidase at 4°C (lane 3). Finally, cells were lysed in 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 60 mM octyl-glucoside for 30 min at 4°C. The extracts were subjected to immunoprecipitation with mAb 9E10 and analyzed by immunoblot with streptavidin-peroxidase to detect the biotinylated protein originally on the surface. (B) COS-7 cells expressing MAL-T were labeled with sulfo-NHS-biotin for 30 min at 4°C, incubated with neuraminidase for 1 h at 4°C, and placed in normal medium at 37°C for 5 min (lane 1), 120 min (lane 2), or 240 min (lane 3). Cells were then lysed and subjected to immunoprecipitation analysis as described in A. The positions of the sialylated (S) and asialo (A) MAL-T species are indicated. The intensity of the bands shown in A and B were quantified by densitometric analysis (see text). SEs were ∼10% of the mean values.