Degradation of internalized Epo and of EpoR. (A) UT-7 cells were preincubated for 15 minutes at 37°C with 10 mM methylamine (▴), 25 μM lactacystin (▪), or vehicles alone (○). 125I-Epo was then added to a final concentration of 1 nM, and the incubation was continued for 30 minutes. The cells were then chilled with 10 vol ice-cold phosphate-buffered saline (PBS) and washed to remove unbound 125I-Epo. Internalized 125I-Epo was determined by acid wash of the cells. For this experiment, this value was 2541 cpm for 10⁶ control cells, 2485 cpm for 106 lactacystin-treated cells, and 2196 cpm for 10⁶methylamine-treated cells. After washing, the cells were further incubated at 37°C with the previously used inhibitors and 50 nM unlabeled Epo to prevent 125I-Epo reassociation with the EpoR. At the indicated times, incubation medium aliquots were sampled, TCA was added to a final concentration of 15%, and the radioactivity of TCA-soluble fractions was measured. (B) Kinetics of EpoR degradation in Epo-stimulated cells. UT-7 cells were preincubated for 15 minutes with cycloheximide alone (control) or in combination with methylamine and/or lactacystin or LLnL as indicated and were stimulated with 10 U/mL Epo for the indicated times. Whole cell extracts were then analyzed by Western blot using C-20 anti-EpoR antibodies. Arrowheads indicate nonspecific bands. (C) Densitometric scanning of the experiment presented in panel B. After scanning, the intensity of band 1 was determined using the ImageJ software. For each inhibitor, the intensity of band 1 at t = 0 was set at 100%, and the intensity of this band at later incubation times was expressed relative to this value.