Fig. 4.

EAPP and R1 repress MAO B promoter by competing with Sp1. A, wild-type (2 kb, construct 1) or deleted (without core promoter, construct 2) MAO B 2-kb luc was cotransfected with Sp1 expression construct together with EAPP (with HA tag) or R1 (with FLAG tag) expression construct into 1242-MG cells. After 24-h incubation, cells were harvested and assayed for luciferase activity. Activity of MAO B promoter-luc cotransfected with the parental empty vector only was set as 100%. B, Western blotting analysis of transfection efficiency of HA-EAPP, FLAG-R1, and Sp1 as used in A. β-Actin was used as a loading control. Representative gels are shown. C, electrophoretic mobility shift analysis showed that Sp1 directly binds to MAO B core promoter-derived Sp1 sites in vitro. The radioactively labeled probe harboring Sp1 sites derived from MAO B core promoter was incubated with various amounts (15, 10, and 5 μg) of purified Sp1 protein as indicated. Nonradioactively labeled probes (lanes 5 and 6) or anti-Sp1 antibody (lane 7) was added into DNA-protein binding reaction when required. Arrows indicate Sp1-DNA complex and shifted Sp1-DNA complex conjugated with anti-Sp1 antibody. A representative gel is shown. D, EAPP and R1 compete with Sp1 for binding to Sp1 sites. The radioactively labeled probe harboring Sp1 sites derived from MAO B core promoter was incubated with purified Sp1 protein in the presence of purified R1 (lanes 1 and 2) or EAPP (lanes 3 and 4) protein. Actin protein was used as a negative control. Arrows indicate R1-DNA, EAPP-DNA, and Sp1-DNA complexes. A representative gel is shown. All data were presented as the mean ± S.D. from at least three independent experiments. *, p < 0.05.