FIGURE 10.

Effect of heme on NCoR1 binding to the FLRev-erbβ·DNA complex. EMSA was utilized to observe the interaction between FLRev-erbβ and FAM-labeled DNA promoter elements. Binding of MBP-NCoR1–540 leads to a concentration-dependent supershift of the Rev-erbβ·DNA complex, which is marginally affected by the addition of Fe³⁺-heme. A, binding assays were prepared in 0.5× TNGD buffer containing 25 ng μl⁻¹ poly(dI-dC), 1 μm FAM-Bmal1 promoter, and the indicated concentrations of FLRev-erbβ_MGC, Fe³⁺-heme, MBP, or MBP-NCoR1–540. Assays were incubated on ice for 2 h prior to separation by native gradient gel electrophoresis. Protein-DNA complexes were imaged using the appropriate excitation and emission wavelengths for fluorescein. B, same as the experiment described for A except FLRev-erbβ_HGS was added (lane orientation is the same for all panels; only protein-DNA complexes are shown for B–D). C, same as described for A except binding assays contained 1 μm FAM-Rev-DR2 instead of FAM-Bmal1 promoter. D, same as described for C except with FLRev-erbβ_HGS.