FIGURE 17.
Heme binding enhances the repressor activity of Rev-erbα but has a negligible effect on Rev-erbβ repression of the Bmal1 promoter. A, HEK293 cells were transfected with 0.1 μg of β-galactosidase control vector, 0.2 μg of pGL3Basic-Bmal1 luciferase reporter, and varying masses of pcDNA3.1(+)-FLRev-erbβ wild-type (wt) or variant expression vectors (indicated below the graph). 24 h after transfection, luciferase activity was measured as described under “Experimental Procedures.” Data are normalized to β-galactosidase activity and are represented as -fold change in luciferase activity based on the reporter control. Three biological replicates were performed with mean ± S.D. shown. B, Western blotting analysis of 50 μg of soluble extracts from a representative replicate experiment described in A, probed with Rev-erbβ QK-6 antibody (top panel), or actin (bottom panel) as a loading control. 5 ng of recombinant FLRev-erbβHGS as a positive control (lane R). A nonspecific reactive band (NS) and a potential Rev-erbβ degradation product (Deg) are indicated with arrows. C, a similar experiment as described in A, except cells were co-transfected with the β-galactosidase control vector, Bmal1 luciferase reporter, and varying masses of pcDNA3.1(+)-Rev-erbα (wt) or the corresponding expression vector for the H602F variant (indicated below graph). Bars represent the average ± S.D. of three biological replicates.