EPOR expression at the cell surface depends sharply upon the history of EPO- exposure.

<p><b>A</b>] Cell surface EPOR detection using antibody EC-c38.5 – Among rabbit monoclonal antibodies generated against an hEPOR ECD, several proved sensitive and specific in flow cytometry assays (Alexafluor 647 second antibody detection). For EC-c38.5, this is illustrated by assays of endogenous EPOR's on UT7epo cells, here following brief EPO withdrawal (8-hours). Negative controls included myeloid HL60 cells, and non-immune rabbit IgG. <b>B</b>] Low ligand levels allow for EPOR expression at the cell surface – In UT7epo cells, cell surface EPOR levels were assayed at 6 hours or 24 hours following EPO withdrawal (and were compared to levels detected during exponential growth in EPO). <b>C</b>] Effects of exposure to EPO at varied concentrations on EPOR internalization also were assessed (at 30 minutes of exposure). <b>D</b>] EPO exposure essentially depletes cell surface EPOR's – UT7epo cells were cultured without EPO for 20 hours. Cell surface EPOR levels were then assayed through time following EPO exposure (3 U/mL). <b>E</b>] Specific detection of cell surface EPOR expression in developing human CD34-derived primary erythroid progenitors – <u>Left panel</u>: Primary erythroid cells were expanded from human BM CD34^pos cells in StemPro34 medium containing SCF (100 ng/mL), IL3 (20 ng/mL), IL6 (20 ng/mL), FLT3 (100 ng/mL), EPO (2.5 U/mL), 0.1 µM beta-estradiol and 1 µM dexamethasone. For cell populations generated at 10.5 days of expansion, and following 12 hours of HGF withdrawal, cells then were exposed (+/−) to EPO for 30 minutes; adjusted to 2°C; and assayed by flow cytometry for levels of cell surface EPOR expression (anti-EPOR antibody EC-c38.5). Myeloid HL-60 cells served as a non-specific binding control. <u>Right panel</u>: Exposure to EPO during HGF withdrawal at doses as low as 0.25 U/mL proved to rapidly down-modulate cell surface EPOR's (for comparison, effects of 15-minute exposure to EPO at 2 U/mL post HGF-withdrawal on EPOR levels were co-analyzed).