Role of the Ser 462 of Epo-R for Epo-R ubiquitination. (A) 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). Cells were lysed using 1% NP40 and lysates were cleared by centrifugation (27,000g for 20 minutes). Lysates were then precipitated with streptavidin and the precipitates were analyzed by Western blot (WB) using anti-Epo-R antibodies. Ubiquitinated forms of Epo-R are indicated (Epo-R/ub). (B) Same experiments as part A; precipitates were analyzed by an anti-ubiquitin antibody (anti-Ub, upper panel) or an anti-Epo-R antibody (C-20, lower panel). Images were recorded using a Fuji Las3000 camera and quantification of the Epo-R was performed using Multigauge V3.0 software (FujiFilm).