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. 1998 Dec 21;188(12):2215–2224. doi: 10.1084/jem.188.12.2215

Figure 6.

Figure 6

Expression of downstream genes depending on Egr-1 activity. (A) Upregulated BP-1 expression in transgenic mice. Bone marrow cells from a BALB/c control littermate and an IA7 mouse were analyzed flow cytometrically by acquiring 3 × 104 live cells stained for B220, IgM, and BP-1. The dot plots are gated B220+ cells and illustrate increased levels and a higher percentage of BP-1 staining in both IgM− and IgM+ subsets. IB10 and ID4 mice showed very similar changes in BP-1 expression. (B) Induction of nur77 in transgenic bone marrow B cells. Using ID4 and BALB/c mice with comparable percentages of mature bone marrow B cells, B220+ cells were purified from the bone marrow as described in the legend to Fig. 2. In the BALB/c sample, protein extracts from 3.3 × 106 B220low and 1.3 × 105 B220high cells (as determined by FACS® analysis) were loaded; for the ID4 sample, the respective amounts were 3 × 106 B220low and 2 × 105 B220high cells. Nur77 expression was detected by Western blotting using a nur77-specific antibody. The amount of protein loaded per lane was controlled by reprobing the blots with a goat anti–mouse IgM antibody. The staining shows elevated nur77 expression in ID4. Size markers (in kD) as indicated were run in parallel to the samples. (C) Binding of recombinant Egr-1 to sequences present in the BP-1 and nur77 promoters. Recombinant Egr-1 was incubated with radioactively labeled oligonucleotides carrying a cognate Egr-1 binding site (lanes 1–7, Egr-1), with an oligonucleotide from the nur77 promoter (lanes 8–14, nur77), or with an oligonucleotide from the BP-1 promoter (lanes 15–21, BP-1) and analyzed by EMSA as described previously (reference 45). The sequences of the respective Egr-1 binding sites are shown (top). Specific binding was proven first by competing either with an excess of an unlabeled oligonucleotide carrying a cognate Egr-1 binding site (lanes 4, 5; 11, 12; 18, 19) or by using an oligonucleotide with an Sp-1 site (lanes 2, 3; 9, 10; 16, 17), and second by inducing a “supershift” by adding the Egr-1–specific antibody C19 to the binding reaction (lanes 6, 13, and 20). Replacing the Egr-1–specific antibody with an Sp-1–specific antibody had no effect on the migration of the DNA–Egr-1 complex (lanes 7, 14, and 21).