c-Myb and Pax-5 synergistically bind the

RAG-2 promoter. (A) Binding of c-Myb and Pax-5 to the −41/−17 region. A radiolabeled −41/−17 oligonucleotide was incubated with various doses of nuclear extract containing either Pax-5, c-Myb, or their combinations, and subjected to EMSA as in Figure 2. Radioactivity of complexes seen in the left gel were measured by BAS2000 and are shown on the right panel. (B) Effect of various c-Myb mutants on the synergistic binding to −41/−17 region with Pax-5. Expression vectors for various c-Myb mutants shown on the top were transfected into 293T cells, and nuclear extracts were prepared 48 hours later. A quantity of 0.1 μL of the nuclear extract containing recombinant c-Myb was incubated with a radiolabeled −41/−17 oligonucleotide in the absence or presence of 0.01 μL 293T cell nuclear extract containing Pax-5, and subjected to EMSA as in Figure 5A (middle panel). Protein concentrations of the nuclear extracts containing c-Myb, c-Myb mutants, or Pax-5 corresponded to those used above. Expression of wild-type or mutant c-Myb in nuclear extracts was verified by Western blot analysis using monoclonal antibody (clone 1.1), which binds to the negative regulation domain of c-Myb (lower panel). ΔNR could not be detected by this antibody. (C) Direct binding of Pax-5 with c-Myb. Expression vectors for both c-Myb and Pax-5 were transfected into 293T cells. After 48 hours, the nuclear extract was prepared and incubated with anti–c-Myb antibody or control antibody. Immunoprecipitates were resolved in SDS-PAGE, transferred to nylon membrane, and probed with anti–Pax-5 antibody or anti–c-Myb antibody. Lysate was applied to SDS-PAGE as a positive control for Western blotting.