Characterization of nuclear extracts made from EPO- and SCF-treated HCD57 (lanes 1 through 4) and HCD57-SREI cells (lanes 5 through 7). Cells (2 × 10⁷) were treated with nothing (lanes 1 and 5), EPO (10 U/mL) (lanes 2 and 6), interferon-γ (5 ng/mL) (lane 4), or SCF (100 ng/mL) (lanes 3 and 6) for 10 minutes at 37°C. The immunoblot was probed with MoAb to STAT5 (A) and reprobed with MoAbs to PY (B). The 95K arrows mark the molecular weight and position where the STAT5 proteins migrate on the gel. (C) Mobility-shift assays of the PIE binding nuclear proteins from EPO and SCF treatment. Nuclear protein preparation and gel shift analyses with the PIE sequence were performed as previously described.11 Nuclear protein (20 μg) from control (C) lanes 1 and 5, and EPO-treated extracts (lanes 2 and 6) and SCF-treated cells (lanes 3 and 7) were incubated with the radiolabeled oligonucleotide, and shifted bands were visualized by autoradiography. Only upon a very dark exposure can a minor band below the position of the major band be seen that arises from STAT1 binding in either EPO- or interferon-treated HCD57 cells. The HCD57 cells seem to be very unresponsive to interferon-γ compared with primary erythroid cells. (D) Super-shift analysis shows that STAT5A/B heterodimers are the constitutively active DNA-binding proteins. The cells were treated as before, or left untreated and the gel mobility shift assay was performed as above except that either anti-STAT1, anti-STAT3, anti-STAT5A, or anti-STAT5B were preincubated for 15 minutes on ice with the nuclear extracts before the addition of the radiolabeled PIE DNA. The bar indicates the supershift of DNA binding that occurred with either anti-STAT5A or anti-STAT5B antisera. The deduced positions of apparent shifts resulting from either STAT5A:STAT5B heterodimers (STAT5A:B), STAT5A homodimers (STAT5A:A), or STAT5B homodimers (STAT5B:B) are indicated by arrows.