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. Author manuscript; available in PMC: 2010 Feb 10.
Published in final edited form as: Curr Biol. 2005 Nov 22;15(22):2044. doi: 10.1016/j.cub.2005.10.053

Figure 2.

Figure 2

Histone deposition by HIR and Asf1/H3/H4 complexes. (A) 30 fmol of a 250 bp radiolabeled DNA probe was incubated with 100 fmol of histones H3/H4 (lanes 2–4), 100 fmol of pre-formed Asf1/H3/H4 complex (lanes 5–9), 100 fmol of Asf1 (lanes 10–12) and either 1.75 μL (lane 6), 3.5 μL (lane 7), or 7 μL (lanes 3, 8, 11, 13) of HIR complex or HIR dialysis buffer (7 μL; lanes 4, 9, 12, 14). Reaction products were resolved on a native 4% polyacrylamide gel and detected by autoradiography. The upper and lower arrows indicate two distinct shifted species. (B) The shifted species contain histone H3. The EMSA was performed as described above, except that 5 μL of HIR complex or HIR dialysis buffer was used. Anti-histone H3 (0.125 μg, Abcam) was added to lanes 6–10. The arrow indicates the supershifted species. (C) Plasmid DNA pre-relaxed with human topoisomerase I was incubated with CBP-TAP purified HIR complex (20 μL) pre-incubated with 0.4 (lanes 2 and 4) and 0.8 (lanes 3 and 5) pmol of histones H3/H4. DNA was analyzed by agarose gel electrophoresis and visualized by SYBR Gold (Molecular Probes) staining.