The association between Epo-R and β-Trcp is induced by Epo and requires Jak2 activity. (A) Consensus β-Trcp recognition motif and conserved β-Trcp recognition motif in Epo-Rs from different species. (B) Specificity of anti Epo-R antibodies. Total cell lysates were prepared from UT-7 cells, OCI-AML-3 cells, parental and Epo-R-transfected BaF3, FDCP-1, or 32D cells. Samples corresponding to 5 × 105 cells were analyzed by Western blot (WB) using SantaCruz C-20 (batch K-200) antibodies. The blot was then stripped and reprobed with anti-actin antibodies to ensure equal loading of the samples. (C) Epo-induced association between the Epo-R and β-Trcp. UT-7 cells were serum- and growth factor-starved for 18 hours and incubated for the indicated time with Epo (10 U/mL). Anti-β-Trcp immunoprecipitates and total cell lysates (TCL) were analyzed using anti-Epo-R antibodies. (D) Effect of Jak2 inhibition on the Epo-R/β-Trcp interaction. UT-7 cells were preincubated with AG490 for 15 minutes before stimulation with Epo for the indicated time. Anti-β-Trcp and anti-Epo-R immunoprecipitates were analyzed using antiphosphotyrosine (anti PY) and anti-Epo-R antibodies. Parts B and C: electrophoresis using C = 1.3% polyacrylamide gels.
