Finer mapping of the SMAD1, hemojuvelin, BMP2, and BMP9 response in the critical BMP-RE2/bZIP/HNF-4/COUP region. Constructs containing mutations in critical distal BMP-RE2/bZIP/COUP/HNF4 region and also the double mutant BMP-RE1/2 were introduced into the 2.5-kb Hamp1 promoter reporter construct as described in Figure 3. Murine 2.5-kb Hamp1 WT was the positive full-length promoter control, 260 bp Hamp1 represented the minimal promoter, and pGL3 represented the negative promoterless control. Control and mutant constructs were transfected into HepG2 cells and either cotransfected with SMAD1 (A) or hemojuvelin (B) or treated with 10 ng/mL BMP4 (C) or BMP9 (D) for 12 to16 hours. Firefly/Renilla ratio of untreated 2.5-kb Hamp1 WT construct was set as 1.00. Means and SEM of the relative firefly/Renilla ratios are shown.