Transcription driven by NF-E2 binding sites is repressed by MCRS2. Transient transfections were performed in 293 cells using HS40-TK-Luc as a reporter. HS40-TK-Luc was constructed by inserting the HS40 element containing NF-E2 binding sites before the HSV-TK promoter of TK-Luc. Briefly, the cells were transiently transfected with luciferase reporter (0.2 μg), β-galactosidase reporter (0.05 μg), and the plasmid constructs expressing HA-Nrf1 (0.2 μg), MCRS2-FLAG (0.4 μg) and β-catenin (S33Y)-FLAG (0.4 μg) as indicated. 36 hours after transfection, the activities of luciferase and β-galactosidase were determined as described under "Experimental procedures". Luciferase activity from transfected cells was normalized with the β-galactosidase activity and expressed as relative luciferase activities compared with the control (lane 1, black bar). Error bars represent the mean ± S.D. from three independent determinations. β-catenin (S33Y)-FLAG in this experiment was used as the control for comparing the effects from MCRS2-FLAG. Other control experiments were also preceded as above, except the reporter was only driven by the HSV-TK promoter (black bar).
Wu et al. BMC Cell Biology 2009 10:9 doi:10.1186/1471-2121-10-9