Fig. 5

The functional EGR1 binding site is located in the distal region of the Adamts-1 promoter. a Western blot analyses of the expression profiles of ADAMTS-1 and EGR1 after transfection of the EGR1 expression vector. GAPDH represents the loading control. b Adamts-1 luciferase vector (− 1705/+ 90) was co-transfected with increasing concentrations of the EGR1 expression vector (pIRES dsRED2/EGR1) in 293 T cells, as indicated. At 48 h post transfection, the cells were collected, and the luciferase activity was measured. c EGR1 expression vector was transiently co-transfected with one of three different Adamts-1 promoter constructs in 293 T cells. The luciferase activity was measured at 48 h post transfection. The firefly activity was normalized to the activity of Renilla luciferase, and the luciferase activity of the untreated cells was designated as one relative value. d EGR1 expression vector was co-transfected in 293 T cells along with one of two different Adamts-1 promoter constructs containing mutations in EGR1 binding sites. The luciferase activity was measured at 48 h post transfection. e Luciferase activity of the WT and mutant (− 1151/− 1134) Adamts-1 promoters co-transfected with an increasing amount of EGR1 expression vector in 293 T cells is indicated. Luciferase activity was calculated relative to the expression of the pGL4.10 basic vector, which served as the negative control. *P < 0.05, **P < 0.01. f Chromatin immunoprecipitation (ChIP)-PCR and real-time ChIP-PCR for four putative EBS in the Adamts-1 promoter. The EGR1-MYC-FLAG expression vector was transfected into 293 T cells and genomic DNA was used for ChIP-PCR. A schematic cartoon (right panel in d) to show locations of four different primers on the Adamts-1 promoter to amplify genomic DNAs precipitated by FLAG antibody. Note that the genomic DNA containing − 1151/− 1134 of the Adamts-1 promoter was significantly enriched. *P < 0.05