FIG. 3.

EMSA of a NF-YB–NF-YC–H3–H4 complex on the Ea promoter DNA. (A) Reconstitution of fragment 2 with increasing amounts of NF-YB4–NF-YC5 alone (200 ng, 400 ng, and 1 μg; lanes 1 to 3), with all core histones (lanes 5 to 7), or with H3-H4 only (lanes 9 to 11). Control reconstitutions with core histones and H3-H4 alone are in lanes 4 and 8. The hybrid complex is indicated. Nuc., nucleosomes. (B) Antibody challenge of the hybrid NF-YB–NF-YC–H3–H4 complex. The hybrid complex was incubated for 30 min at 4°C with increasing amounts of anti-NF-YB (100 and 500 ng; lanes 2 and 3), antihistone (10 ng, 100 ng, and 1 μg; lanes 5 to 7), or control anti-Gata1 (100 and 500 ng; lanes 9 and 10) antibodies (Ab). The supershifted complexes are indicated by asterisks. (C) The indicated combinations of proteins (H2A-H2B [lanes 1 and 6], H3-H4 [lanes 2 and 7], H3–H4–NF-YB–NF-YC [lanes 3 and 8], H2A-H2B-H3-H4 [lanes 4 and 9], and H2A-H2B-H3-H4-NF-YB-NF-YC [lanes 5 and 10]) were incubated with wt Ea DNA (lanes 1 to 5) or with a Y-box mutant (lanes 6 to 10). (D) Same as panel C except that H3-H4 tetramers were incubated alone (lanes 1 and 5) or with NF-YB4-NF-YC5 (200 ng [lanes 2 and 6] and 1 μg [lanes 3 and 7]). Nucleosomes are in lanes 4 and 8.