Figure 2.

Sp3 does not form oligomeric complexes on promoters containing Sp-binding sites. (A) Complex formation of Sp3 with ³²P-labeled DNA probes containing either one (BCAT-1) or two (BCAT-2) Sp-binding sites. Increasing amounts of affinity purified Sp3 (1.2, 2.4, 5.9 and 18 ng protein) were added to the reaction mixtures (20 µl) containing 0.2 ng of BCAT-1 (lanes 1–4) or BCAT-2 probes (lanes 5–9). After 20 min of incubation at room temperature, 7.5 µl of the reaction mixture was separated on a native polyacrylamide (4%) gel as described in Materials and Methods. The reaction mixtures in lanes 10–12 contained 10 ng of Sp3. The reaction mixture in lane 10 has no antibody whereas the reaction mixture in lane 11 received 0.1 µg of Sp3-antibody and reaction mixture in lane 12 received 0.1 µg of Sp1-antibody. (B) Complex formation of affinity purified Sp1 with ³²P-labeled DNA probes containing either one (BCAT-1) or two (BCAT-2) Sp-binding sites. Increasing amounts of Sp1 (0.8, 1.6, 3.2 and 7 ng protein) were added to the reaction mixtures (20 µl) containing 0.2 ng of BCAT-1 or BCAT-2 probes. After 20 min incubation at room temperature, 7.5 µl of the reaction mixture was separated on a native polyacrylamide (4%) gel as described in Materials and Methods. The reaction mixtures in lanes 9–11 contained 5 ng of Sp1. The reaction mixture in lane 9 has no antibody whereas the reaction mixture in lane 10 received 0.1 µg of Sp1 antibody and the reaction mixture in lane 11 received 0.1 µg of Sp3 antibody.