Inhibition of surface expression of TfR1, SCFR, EPOR, IL-3R, and of total protein of TfR1 and GATA-1 in erythroid cells cocultivated with HZ or treated with HNE. Erythroid cells were cocultivated with HZ (25μM HZ-heme), treated with HNE (7μM), or kept as untreated controls (CTRL) in liquid culture. (D) Erythroid cells were cocultivated with unfed (Mo), HZ-fed (Mo + HZ), or RBC-fed (Mo + RBC) monocytes in liquid culture. (A-B,D) The surface expression of antigens was quantified by flow cytometry after immune staining and expressed in panel A as the percentage of TfR1 highly positive treated cells referred to highly positive control cells and in panels B and D as relative MFI (REL MFI). Relative MFI is the ratio of the MFIs of treated to untreated cells. Means ± SE of 3 or more independent experiments. The significances of differences (P < .05) between controls and HZ-treated (*) or HNE-treated (§) cells (A-B), or between erythroid cells cocultivated with HZ-fed monocytes and unfed (*) or RBC-fed (§) monocytes are indicated (D). (C-E) Total protein expression of TfR1 (C) and GATA-1 (E) was analyzed in 15 μg and 30 μg lysate protein from cells harvested on days 1 and 6 after addition of HZ/HNE. Proteins were separated by 1-dimensional SDS-PAGE and Western blotted. Transferred proteins were probed with specific primary antibodies and detected by binding of appropriate secondary antibodies. TfR1 and GATA-1 evidenced by ECL and the densitometric analysis are shown. One representative experiment of 3 is shown. Actin is included as loading control. (F) Representative expression profiles of cell surface TfR1, SCFR, EPOR, and IL-3R measured by flow cytometry in erythroid cells after 1 day coincubation with HZ (solid line filled space) and HNE (dotted line unfilled space); control erythroid cells plotted as solid line/unfilled space and background as dashed line/unfilled space.