Fig. 3.
EAPP and R1 repress MAO B promoter and catalytic activities. A, Wild-type (2 kb, construct 1) or deleted (without core promoter, construct 2) MAO B 2-kb luc was cotransfected with EAPP or R1 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 was set as 100%. B, 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) used in ChIP assay. C, 1242-MG cells were subjected to ChIP assay using anti-EAPP, anti-R1, or anti-Sp1 antibody followed by real-time PCR with primers specific for MAO B core promoter. PCR with primers targeting 5′-irrelevant locus was used as a negative control. The occupancy by EAPP, R1, and Sp1 at the core promoter was quantitated using the 2−ΔΔCT method and presented as the percentage of input. D, MAO B catalytic activity was determined in 1242-MG cells that stably overexpress EAPP, R1, or Sp1. 1242-MG cells stably transfected with the parental empty vector was used as a control. All constructs carry neomycin-resistant genes. All data were presented as the mean ± S.D. from at least three independent experiments. *, p < 0.05.