Fig. 5.
EAPP, R1, and Sp1 cooperatively regulate glucocorticoid activation of MAO B promoter. A, wild-type (2 kb, left) or deleted (without core promoter, right) MAO B 2-kb luc was cotransfected with GR expression construct together with EAPP, R1, or Sp1 expression construct into 1242-MG cells. Cells were then treated with 100 nM dexamethasone for 24 h followed by luciferase activity determination. Ethanol was used as a vehicle. Activity of MAO B promoter-luc cotransfected with GR expression construct only under no treatment was set as 1. B, Western blotting analysis of transfection efficiency of HA-EAPP, R1, Sp1, and GR as used in A. β-Actin was used as a loading control. Representative gels are shown. C, MAO B 2-kb luc was cotransfected with Sp1 expression construct together with various amounts of EAPP (left) or R1 (right) expression construct into 1242-MG cells. Cells were then treated with 100 nM dexamethasone for 24 h followed by luciferase activity determination. Ethanol was used as a vehicle. Activity of MAO B 2 kb-luc transfected with the parental empty vector only under no treatment was set as 1. D, a schematic diagram of MAO B 2-kb promoter showing the location of core promoter region that encompasses Sp1 sites (−287/−153) and 5′-irrelevant locus (−1940/−1780) as used in ChIP assay. 1242-MG cells treated with or without dexamethasone (100 nM, 24 h, ethanol used as a vehicle) were subjected to ChIP assay using anti-EAPP, anti-R1, or anti-Sp1 antibody followed by real-time PCR with primers specific for the core promoter region. The occupancy by EAPP, R1, and Sp1 at specific locus was quantitated using the 2−ΔΔCT method and presented as the percentage of input. All data were presented as the mean ± S.D. from at least three independent experiments. *, p < 0.05; **, p < 0.01.