c-Myb with Pax-5 binds −41/−17 region of the

RAG-2 promoter. (A) DNA sequence of the mouseRAG-2 promoter region of −65 to +4. Putative binding sites for Pax-5 and c-Myb (c-Myb1 and c-Myb2) are shown by arrows under the sequence. Nucleotides used for c-Myb binding site mutation are indicated above the sequence. Nucleotides conserved between humans and mice are shown by capital letters. +1 indicates major transcription initiation site. (B) DNA-sepharose precipitation assay. Nuclear extracts were prepared from 293T cells transfected with pAct vector (Mock) or pAct–c-Myb (r-c-Myb), or from 18.8.1 cells (top panel) or 293T cells transfected with pEF-BOS vector (Mock) or pEFBOS–Pax-5 (r-Pax-5), or 18.8.1 cells (bottom panel). Extracts were incubated with sepharose beads conjugated with either oligonucleotides containing the STAT3 binding site (control) or −41/−17. Bound proteins were eluted and resolved in SDS-PAGE and detected by immunoblotting using c-Myb or Pax-5 antibody. (C) Binding of c-Myb and Pax-5 in nuclear extracts from 18.8.1 cells to the −41/−17 region. Nuclear extracts were prepared from 18.8.1 cells or 293T cells transfected with either pAct–c-Myb (r-c-Myb) or pEFBOS–Pax-5 (r-Pax-5) and subjected to EMSA with a radiolabeled −41/−17 oligonucleotide. Oligonucleotide competition was carried out with 200-fold excess of unlabeled −42/−17 oligonucleotide, the oligonucleotide containing the consensus c-Myb binding sequence (c-Myb) or its mutant binding sequence (c-Mybm), the oligonucleotide containing the consensus Pax-5 binding sequence (Pax-5) or its mutant binding sequence (Pax-5m), or the oligonucleotide containing the consensus GATA binding sequence (GATA). For supershift assay, EMSA was performed in the presence of 0.5 μg polyclonal Pax-5 antibody or control IgG. Specific protein/DNA complex is indicated as C and free probe as F. Supershifted band is indicated by an asterisk.