Elimination DNA-binding activity of extracts by coincubation with differentiated cell extracts. (A) Twelve micrograms of HL-60 extract was incubated in the absence (−) or presence of 12 μg of extract from CD34⁺ blast cells (0) or CD34⁺ cells incubated for 3 to 15 days in the presence of kit ligand (100 ng/mL), IL-3 (100 ng/mL), and G-CSF (20 ng/mL) as indicated. After 20 minutes of coincubation, radiolabeled target DNA was added and gel-shift was performed as described. (B) (Left half) Thirty micrograms of extract from HL-60 cells (H) and cord neutrophils (N) were incubated separately or together (H+N) before the addition of labeled 44-bp fragment or labeled SIE probes. The typical gel-shift band seen upon incubation with each fragment is indicated with an arrow (forward arrow, 44-bp band shift; backward arrow, SIF bands). The lower, nonspecific band seen in the H+N lane was not extinguished by unlabeled competitor 44-bp fragment (not shown). (Right half) Twenty-six micrograms of HL-60 extract (H) or 9 μg of extract from sorted CD11b⁻CD15⁺ cells (CD15) or from CD11B⁺CD15⁺ cells (11b/15) were incubated separately with labeled 44-bp fragment as shown. In addition, these amounts of extracts were combined in mixing experiments (H+CD15), (H+11b/15) as indicated. Specific bands are indicated as before. (C) Incubation of differentiating cell extracts with SIE. Extracts from CD34⁺ cells incubated for 3 to 15 days in the presence of kit ligand, IL-3, and G-CSF were incubated with radiolabeled SIE target. Characteristic bands (SIF⁴³) are evident.