Web Fig 2.Confirmation of BCL-6 as a Blimp-1 Target. (A) Expression of BCL-6 mRNA assessed by RT-PCR in BJAB and RAJI cells infected with control or Blimp-1-expressing virus. GAPDH expression serves as the control. (B) Western blot of BCL-6 protein in BJAB and RAJI cells infected with control or Blimp-1-bearing virus. SP1 expression is used as a control. (C) A gel mobility shift assay detects abundant, specific BCL-6 DNA binding activity in RAJI cells carrying an empty inducible vector (pMEP4). Specificity is demonstrated by competition with cold competitor oligos and BCL-6-specific anti-sera. DNA binding activity is abolished in RAJI cells expressing doubly-inducible Blimp-1-ERD after induction with cadmium (Cd) and tamoxifen (TMX). Expression of Blimp-1-ERD was also confirmed in these cells by gel shift assay (data not shown). (D) Gel shift demonstrating Blimp-1 binding to a conserved site in BCL-6 intron-1. EMSA was performed as described using P3X nuclear extract with the mouse c-mycBlimp-1 binding site. Specificity is shown by competition with excess c-myc and non-specific oligos. The putative Blimp-1 site in intron-1 of human and mouse BCL-6 competes effectively for Blimp-1 binding. The sequence of this conserved site (with nucleotide positions) is shown with consensus, overlapping Blimp-1 sites underlined.