C/EBPβ binds to the promoter regions of IRF4, of BLIMP1, and in exon 1 of BCL2. (A) mRNA expression of C/EBPβ, IRF4, BCL2, and BLIMP1 in H929 MM cells transfected with EV, WT-C/EBPβ, or DN-C/EBPβ were analyzed by quantitative RT-PCR. Data were analyzed according to the ΔCT method. The results are expressed as mRNA fold change compared with control pcDNA group. (B) Chromatin immunoprecipitation with anti-C/EBPβ antibody and RT-PCR on the resulting genomic DNA was conducted on H929 MM cells. PCR probes to the various promoters are indicated and described further in “RT-PCR analysis.” The a and b probes for IRF4, BLIMP1, and BCL2 represent 2 areas of CAAT sequence in the promoter regions. The C/EBP and CCR5 probes were to intron regions previously shown to bind the protein. DNA that was immunoprecipitated by RNA polymerase II was used as the standard (set to 100%) and the amount of DNA immunoprecipitated by C/EBPβ antibody was normalized to it. Control represents the amount of DNA immunoprecipitated by normal rabbit IgG (a negative control). Error bars represent the SD of the mean from at least 4 determinations.
