Figure 4.

Trafficking of newly synthesized CD through the MVBs. To measure the rate of appearance of newly synthesized CD in MVBs, HEp-2 cells were 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 being transferred to 37°C for up to 30 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). Results are means ± SE of three observations.