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. 2003 Aug;23(16):5857–5866. doi: 10.1128/MCB.23.16.5857-5866.2003

FIG. 5.

FIG. 5.

(A) Western blot showing the protein levels of wild-type Hap1 and Hap1 mutants in yeast cell extracts. Extracts were prepared from yeast cells expressing wild-type Hap1 (lane 2), smHap1 (lane 1), H7-dd1 (lane 3), H7-dd2 (lane 4), H7-dd3 (lane 5), H7-dd4 (lane 6), or H7-dd5 (lane 7). Approximately 30 μg of total proteins from each extract was loaded onto 7.5% SDS-polyacrylamide gels, and the proteins were electrophoresed, transferred to PVDF membranes, and probed with an anti-Hap1 antibody (22). Hap1 proteins were visualized by using a chemiluminescence Western blotting kit (Boehringer Mannheim). (B) DNA-bound complexes formed by wild-type Hap1 and mutants. Approximately 20 μg of protein extracted from cells expressing wild-type Hap1 (lanes 1 and 2), smHap1 (lanes 3 and 4), or H7-dd3 (lanes 5 and 6) was incubated with radiolabeled DNA in the absence (lanes 1, 3, and 5) or presence of 2 (lanes 2, 4, 6, and 7), 5 (lane 8), or 10 (lane 9) μg of heme per ml. The reaction mixtures were loaded onto 3.5% nondenaturing polyacrylamide gels and electrophoresed, and the bands were visualized by using a PhosphorImager. These experiments were repeated at least twice.