Nonerythroid cells express low to undetectable levels of EpoR. (A) EPOR mRNA was quantified in hematopoietic and nonhematopoietic cells using a publicly available microarray from the Gene Expression Omnibus (www.ncbi.nih.gov/geo) with mean fluorescence units plus or minus SEM for probe 215054 presented. Data were normalized using global mean scaling. Similar findings were also observed with other EPOR probes (data not shown). PB indicates peripheral blood; BM, bone marrow. (B) Levels of EPOR transcripts were quantified by quantitative RT-PCR in positive control UT-7/Epo cells (+), negative/low control 769-P cells (−), primary human endothelial cells, and normal human nonhematopoietic tissues using primers located in exons 5 and 8. Results were normalized to levels of cyclophilin A mRNA. Data are mean ± SEM (n = 3 per set) and are representative of 3 separate experiments. These findings were reproduced with a different EPOR primers in exon 8 (data not shown). (C) EpoR protein expression analysis in endothelial cells using immunoblot (IB) analysis with anti-EpoR monoclonal antibody A82 and cyclophilin B as a loading control (experiment performed twice). Positive controls FLAG-EpoR transfected into COS-7 cells, UT-7/Epo, and OCIM-1 cells were used to determine the position of EpoR on the blot. FL indicates full-length EpoR at 59 kDa. The smaller proteins detected in erythroid samples were confirmed to contain EpoR sequences and probably reflect degradation products.^3,22  (D) Surface EpoR was determined using [¹²⁵I]-rHuEpo binding. Data are mean ± SEM (n = 5 per cell type) and are representative of 3 experiments. Inset with expanded axis represents absence of specific binding of rHuEpo to endothelial cells.