c-Myb activates the RAG-2 promoter by binding the c-Myb1 site in the promoter region.

(A) Promoter activity of wild-type versus c-Myb binding site–mutatedRAG-2 promoter. A quantity of 10 μg luciferase constructs linked to the −86/+147 promoter fragment or that with mutation in c-Myb binding sites (c-Myb1m or c-Myb2m) together with pSRα-LacZ was transfected into 18.8.1 cells or BAL17 cells, and luciferase activity was analyzed as in Figure 1. Error bars indicate deviation of 3 experiments. (B) Effect of mutation in c-Myb1 site on binding of recombinant c-Myb and Pax-5 to the −41/−17 region. Nuclear extracts were prepared 48 hours after transient transfection of 293T cells with pAct vector (Mock), pAct–c-Myb (r-c-Myb), or pEFBOS–Pax-5 (r-Pax-5) DNA and subjected to EMSA as in Figure 2C. To verify specific binding, 50-fold excess of unlabeled −41/−17 oligonucleotide (WT), or 50-fold or 200-fold excess of a similar oligonucleotide containing mutations at the c-Myb1 site (c-Myb1m) was included in EMSA. Specific DNA/protein complex is indicated as C1 (for c-Myb) and C2 (for Pax-5), and free probe as F. Two bands are detected for c-myb (C1), probably reflecting the monomer (lower band) and dimer (upper band) formation of c-Myb protein(s) in the gel Pax-5.29