BSAP factor identification by specific binding site mutations and supershift EMSA. (A) Sequence comparison between the BSAP consensus-binding site and the wild-type fragment (+ 28 + 59) or mutated fragment (+ 28 + 59^mut) sequences corresponding to abolish the possible BSAP-binding site. Bold capital letters represent the most conserved nucleotides. Capital and lowercase letters represent the nucleotides matched and unmatched to the consensus-binding site, respectively. Underlined letters are the mutated ones. (B) EMSA with Daudi nuclear extracts and the fragment (+ 28 + 59) used as probe (lane 1). Competition experiments were performed with the cold wild-type fragment (+ 28 + 59) (lanes 2-4) or mutated fragment (+ 28 + 59^mut) (lanes 5,6). Molar ratios of the cold fragment were 100 × (lanes 2,5), 50 × (lanes 3,6), and 10 × (lanes 4,7) (C) Supershift experiment with Daudi nuclear extract and probe + 1/+ 104. (Lanes 1 and 2) Probe without and with extracts, respectively. Antibody against BSAP (1 μL) was added to the assays (lane 3). A nonspecific antibody control (1 μg/μL) was used (lane 4). (D) Same as panel C, except probe + 28 + 59 was used and the antibody against BSAP was added to the assays in descending concentration (0.1, 0.05, and 0.01 μL) from lane 3 to lane 5. Control nonspecific antibody (lane 6).