Figure 6. Identification of C1 and C3 binding activity as Sp1 and Sp3, respectively. (A) An electrophoretic mobility shift assay (EMSA) was performed with ³²P-labeled probes covering the promoter fragment -53/-24 and 8 µg nuclear extract prepared from MA-10 cells or 2 µl recombinant Sp1 proteins. Mouse monoclonal antibody against a peptide consisting of amino acids 520-538 of Sp1 was added to the EMSA reaction. (B) EMSA was performed with various promoter fragments (-723/-693, -53/-24, and -86/-50) and 8 µg nuclear extract. Mouse monoclonal antibody against Sp1 and rabbit polyclonal antibody against the carboxy terminus of Sp3 or of Egr-2 were added to the EMSA reaction as indicated above each lane. (C) EMSA was performed with the promoter fragment -723/-693. Competitions were carried out by adding 2500 fmol of unlabeled consensus motifs for the transcription factors Egr and AP2 to the EMSA reactions. Specific DNA/protein complexes are designated C1, C2, C3, C4, C5, and C6-9 on the left. ^* refers to the super-shifted band.