Figure 7.

Effect of MG-132 on transferrin recycling and degradation of ¹²⁵I-RAP. (A) ts20 cells were serum depleted for 1 h with or without 20 μM MG-132 and then loaded with 2 μg/ml ¹²⁵I-Tf for 30 min at 30°C. Cells were chilled on ice, plasma membrane-bound ¹²⁵I-Tf was removed, and the cells were incubated at 30°C in medium containing 50 μM desferal in the absence or presence of MG-132. The release of ¹²⁵I-Tf was determined and expressed as a percentage of the total amount of radioactivity loaded in the cells. ●, control; □, MG-132. (B) LRP-null CHO cells stably transfected with mLRP4T100 were incubated for 1 h at 37°C with or without 20 μM MG-132 before 5 nM ¹²⁵I-RAP was added. The incubation was continued for 6 min, after which the unbound radioactivity was removed and the cells were incubated at 37°C in the absence of ligand with or without MG-132 for the time points indicated. At each time point the amount of cell surface, internalized, and degraded ligand was determined as explained in MATERIALS AND METHODS. The amount of ¹²⁵I-RAP is plotted as a percentage of total radioactivity. Each point in the graph represents the mean value of two experiments performed in duplicate ± SD. (Left) Intracellular ¹²⁵I-RAP. (Right) Degraded ¹²⁵I-RAP in the medium. ●, control; □ MG-132.