Bach2 is a heme-binding protein. (A) A schematic representation of the Bach1 and Bach2 domain structure. The bZip domain, BTB domain (Broad complex-Tramtrack-Bric-a-brac domain), known as a protein-protein interaction motif, and CP motifs are indicated with boxes and filled circles. GST-Bach2 and GST-Bach2mCP illustrate the recombinant Bach2 fragments examined in this study. GST-Bach2mCP contains Cys→Ala mutations in the 5 CP motifs. (B) The heme titration of GST-Bach2 (1μM) was monitored by the differences in absorption spectra. Each arrow represents the directions of the absorbance changes with increasing heme concentrations. (C) The titration curve observed at 401 nm for GST-Bach2 with heme. (D) The heme titration of GST-Bach2mCP as monitored by the differences in the absorption spectra. (E) EMSA was performed by the use of 60 ng of each recombinant GST-Bach2 and a MARE DNA probe. The heme concentrations were 0.05μM (lane 3), 0.1μM (lane 4), 1μM (lane 5), 3μM (lane 6), and 10μM (lane7). Representative EMSA data are shown from 3 independent experiments. (F) EMSA was carried out with 50 ng of recombinant MBP-MafK. The heme concentrations were the same as in panel E. Representative EMSA data are shown from 2 independent experiments. (G) The band intensities in panels E and F were measured by a densitometry analysis. The open circle with a dashed line, closed circle with a line, and closed gray circle with a gray line indicate 3 independent experiments of EMSA with GST-Bach2 proteins. The open box with the dashed line and filled box with the line indicate 2 independent EMSA experiment with MBP-MafK proteins.