Degradation kinetics of the EpoR in Epo-starved cells and Epo-stimulated cells. (A) Characterization of the EpoR forms. (i) EpoR immunoprecipitates from Epo-starved UT-7 cells were incubated for 18 hours at 37°C with endoglycosidase H (Endo H), endoglycosidase F (Endo F), or deglycosylation buffer alone (control) as previously described.29 (ii) Whole UT-7 cells were incubated for 45 minutes at 4°C without or with 100 μg/mL proteinase K (Prot K), then were washed, and solubilized in electrophoresis sample buffer. Proteins were then analyzed by Western blot using C-20 anti-EpoR antibodies. (B) Stability of the EpoR proteins in Epo-starved cells. UT-7 cells were incubated with cycloheximide to block protein synthesis. Cells were sampled at the indicated times, and whole cell extracts were analyzed by Western blot using C-20 anti-EpoR or anti-Jak2 antibodies. (C) Stability of the EpoR proteins in Epo-stimulated cells. UT-7 cells were preincubated with cycloheximide for 15 minutes. Cells were then stimulated with 10 U/mL Epo for the indicated times, and whole cell extracts were analyzed by Western blot using C-20 anti-EpoR (top) or anti-Jak2 (bottom) antibodies. The number 1 indicates mature form of the EpoR; IgG, the heavy chains of the immunoprecipitating antibodies; 2, maturing form of the EpoR; 3, deglycosylated EpoR; and arrowhead, nonspecific bands.