Binding of c-myb to myb binding sites in the cyclin A1 promoter (EMSA). (A) Expression of c-myb in nuclear extracts of Cos-7 cells either transfected with empty vector (Cos) or with a c-myb expression vector (Cos-myb). Western blot analysis was performed similar to the experiment shown in Fig 3. (B) Binding of the different nuclear extracts to a c-myb consensus binding site and to the cyclin A1 promoter myb binding site at position +2. The nuclear extract containing c-myb protein led to a c-myb containing band at the consensus site as well as at the cyclin A1 promoter myb site. This band was successfully competed away by a 50-fold excess of the nonlabeled oligonucleotide encompassing the cyclin A1 myb site but not by a 50-fold excess of a nonspecific oligonucleotide. Also, the addition of 2 μg of a murine isotype control antibody did not alter the appearance of the band. However, an anti–c-myb antibody led to a supershift of the band.