Importance of the Ser 462 of Epo-R for interaction with β-Trcp. (A) Expression of Epo-R in UT-7 cells and in Epo-R-transfected BaF3 cells. Total cell lysates prepared from UT-7 cells, untransfected BaF3 cells, and BaF3 Epo-R^WT cells no. 1 and no. 2, BaF3 Epo-R^S462Acells no. 1 and no. 2, were analyzed by Western blot (WB) using a C = 1.3% polyacrylamide gel and anti-Epo-R antibodies. The blot was then stripped and reprobed with anti-actin antibodies to ensure equal loading of the samples. (B) Cell surface Epo-R is quantitatively precipitated by streptavidin after bEpo binding. Starved UT-7 cells were stimulated for 10 minutes with 10 U/mL bEpo. Cells were lysed using NP40 1% and lysates were cleared by centrifugation (27,000 g, 20 minutes). Clarified lysates were then precipitated with streptavidin beads. Extracts before streptavidin precipitation (TCL), unbound material, and material precipitated by streptavidin, each corresponding to 5 × 10⁵ cells, were analyzed by Western blot using anti-Epo-R antibodies. (C) Ser 462 of Epo-R is required for β-Trcp binding. After Epo deprivation as described in “Materials and methods,” UT7, BaF3 Epo-R^WT, and BaF3 Epo-R^S462A cells were stimulated or not with biotinylated Epo (bEpo). Clarified lysates were precipitated with streptavidin beads and the precipitates were analyzed by Western blot using anti-β-Trcp and anti-Epo R antibodies.