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. 2002 Jul;22(13):4771–4780. doi: 10.1128/MCB.22.13.4771-4780.2002

FIG. 2.

FIG. 2.

FIG. 2.

FIG. 2.

Blimp-1 binds to the Pax-5 promoter and represses Pax-5 promoter activity. (A) The alignment of the putative Blimp-1 binding site in the murine Pax-5 promoter with known sites in the murine c-myc promoter, human CIITA promoter III, and human IFN-β promoter. (B) EMSA with nuclear extracts from P3X plasmacytoma and 18-81 pre-B cells and a 30-bp probe derived from the putative Blimp-1 site in the murine Pax-5 promoter. Oligonucleotide B contains the Blimp-1 site in the murine Pax-5 promoter. Oligonucleotide N is a nonspecific oligonucleotide. pre, preimmune serum; Bl, Blimp-1-specific polyclonal antiserum. The molar excess of cold competitors is indicated. The arrow indicates the specific binding complex. (C) Comparison of the binding affinity of Blimp-1 to sites in the mouse c-myc promoter and mouse Pax-5 promoter. EMSA was performed with nuclear extracts from P3X and a probe containing the Blimp-1 binding site in the mouse c-myc promoter. Oligonucleotide P contains the Blimp-1 site in the mouse c-myc promoter. Oligonucleotide B contains the Blimp-1 site in the mouse Pax-5 promoter. N is a nonspecific oligonucleotide. The molar excess of cold competitors is indicated. N.S., nonspecific binding complex. (D) 3T3 cells were cotransfected with different amounts of Blimp-1 expression vectors, as indicated in the figure, and 2 μg of luciferase reporter driven by a 1.8-kb region of the mouse Pax-5 promoter (BSAP-Luc), the Pax-5 promoter with a mutated Blimp-1 binding site (M BSAP-Luc), or CIITA promoter III (CIITA-Luc). The relative luciferase activity represents the normalized luciferase activity derived from cells transfected with the Blimp-1 expression vector (pBDP1-F) divided by the normal-ized luciferase activity derived fromcells transfected with a control vector (pBDP1-B). The luciferase light units from one representative experiment were BSAP-Luc plus 2 μg of control vector 700137 and MBSAP-Luc plus 2 μg of control vector 548658. Averages ± the standard deviations from four independent experiments are shown. Diagrams of BSAP-Luc and M BSAP-Luc with the Blimp-1 binding sequence and mutated sequence and their relative distance to the transcription start sites in the Pax-5 promoter are indicated. The hatched box represents the Pax-5 gene sequence, and the arrow indicates the transcription start site. (E) Raji cells were cotransfected with 5 μg of Blimp-1 expression plasmid and 10 μg of luciferase reporter driven by the mouse Pax-5 promoter (WT) or with a mutated Blimp-1 site (M). The relative luciferase activity was determined as described in panel D. The luciferase light units from one representative experiment were the wild type plus the control expression vector 36840 and the mutant plus the control expression vector 34650.