Figure 4.

Mafb directly regulates Nephrin expression. (A) Analysis of the 5′-flanking region of Nephrin by the Ensembl Genome Database Web site identified an Mafb binding site (half-MARE) that is highly conserved between mouse and human (Nephrin-MARE). (B) Reporter assay. WT Nephrin-MARE (WT-Luc) and mutated Nephrin-MARE (Mut-Luc) luciferase reporter constructs are indicated. The 293T cells were cotransfected with the indicated Nephrin promoter–reporter plasmid constructs along with the Mafb expression plasmid, and the relative luciferase activity was measured as described in Concise Methods. The relative luciferase activity of the reporter plasmids is shown in the lower panel with the activity generated from cells transfected with the empty reporter vector (0 ng) and the Mafb expression plasmid defined as 1.0. Each bar represents the mean±SEM. **P<0.05. (C) EMSA using labeled consensus MARE (upper panel) and Nephrin-MARE (lower panel). (Lane 1) Biotin-labeled consensus MARE probe. (Lane 2) Mafb protein and biotin-labeled consensus MARE probe. (Lane 3) Lane 2+anti-Mafb antibody. (Lane 4) Lane 2+unlabeled consensus MARE oligonucleotide. (Lane 5) Lane 2+unlabeled Nephrin-MARE oligonucleotide.