Figure 5.
The effect of tunicamycin on trafficking of CD through MVBs. Cells were preincubated with tunicamycin (10 μg/ml) for 2 h at 37°C, and then pulsed with 35S-labeled amino acids for 15 min at 37°C, washed, and incubated for 20 min at 37°C and then maintained at 20°C for 4 h. Anti-TR-gold was added for the last hour at 20°C before transfer to 37°C for up to 60 min in the continued presence of anti-TR-gold. Gold-loaded MVBs were then isolated as described in MATERIALS AND METHODS. The amount of newly synthesized CD in the isolated MVB fractions was determined by immunoprecipitation and expressed as a percentage of the total loaded on the fractionation gradient (% PNS).