Degradation of the intracellular domain of the EpoR by proteasomes.(Left panel) UT-7 cells were incubated for 30 minutes with 125I-Epo. After washing to remove unbound radioactivity, the cells were lysed, and clarified cell extracts were cross-linked with 2 mM BS3. Excess cross-linking reagent was blocked with ethanolamine, and EpoRs (ER) were precipitated with a polyclonal antibody directed against the intracellular domain of the receptor (C-236). Immunoprecipitates (IP) were dissociated by boiling in SDS- and DTT-containing buffer. Parts of the eluted proteins were analyzed (lane 1). The remaining proteins, after removal of DTT and dilution in Nonidet P-40 (NP-40)–containing buffer, were reprecipitated successively with anti-GST (lane 2), C-236 anti-EpoR (lane 3), and anti-Epo (lane 4) antibodies. Immunoprecipitates were analyzed by polyacrylamide gel electrophoresis and autoradiography. (Right panel) UT-7 cells were preincubated for 15 minutes with no inhibitor (control), LLnL, or methylamine (MetAm) and stimulated for 30 minutes with125I-Epo. EpoR immunoprecipitates of cross-linked cell extracts were prepared as in left panel. Denatured immunoprecipitates were then immunoprecipitated with C-236 anti-EpoR antibodies and analyzed by polyacrylamide gel electrophoresis and autoradiography. Arrows A and B point to the 70 kDa and 40 kDa EpoR crosslinked to125I-Epo.
