FIG. 6.

Binding of Pax5 to Ebf1β regulatory sequences in vivo. (A) Schematic representation of the Ebf1β promoter and regions analyzed by ChIP. (B) ChIP was performed in the indicated cell lines with anti-Pax5 and the control IgG antibody. Enrichment (n-fold) comparing immunoprecipitate with the specific anti-Pax5 antibody and the control IgG was determined by real-time PCR; n.d., not detected. (C) Semiquantitative PCR was performed with serial dilutions of template DNA from cultured bone marrow pro-B cells of wild-type (wt) and Pax5^−/− mice. Binding can be detected with anti-Pax5 antibodies but not with nonspecific IgG on both the Ebf1β and CD19 promoters. The anti-Pax5 antibodies do not bind to the JunB promoter. (D) Transient transfection of Ba/F3 cells with empty vector only or with 3 μg Pax5 and 3 μg Ets1 followed by quantitative RT-PCR of endogenous Ebf1α, Ebf1β, or total Ebf1 mRNA. mRNA levels were normalized to β-actin, and the expression level of the empty-vector control was set to 1. Error bars represent the standard deviations of the means of three experiments. (E) IL-7-cultured pro-B cells of Pax5-deficient mice have reduced EBF1 protein levels. Intracellular staining of IL-7-cultured pro-B cells was performed with monoclonal anti-EBF1 antibody and secondary anti-rat-FITC antibody. For the control staining only secondary antibody was used. (F) Fraction B pro-B cells of mice deficient for Pax5 show a reduction of Ebf1β mRNA and total Ebf1 mRNA. Real-time RT-PCR analysis was performed with sorted fraction B cells of wild-type (wt) and Pax5^−/− mouse fetal liver (B220⁺ CD43⁺ HSA⁺ BP-1⁻). Ebf1α, Ebf1β, and total Ebf1 mRNAs have been amplified by oligonucleotides specific for exon 1a, exon 1b, or both isoforms. As a control, RT-PCR of B29, mb1, and λ5 was performed. mRNA levels were normalized to β-actin, and the expression level of the wild type was set to 100%. Error bars represent the standard deviations of the means of three experiments.