FIGURE 6.

GR binds to the GRE-like sequences in the human CALD1 promoter in vivo and in vitro. A, ChIP assay of A549 cells treated with vehicle or 1 μm DEX for 2 h. The cell extracts were subjected to ChIP using the anti-GR antibody. The DNA fragments of the CALD1 promoter region containing the two GRE-like sequences were amplified by PCR. The SGK1 and GAPDH promoter regions were used as the positive and negative controls, respectively. B, DNA-binding assay of GR to GRE-like sequences. Each biotinylated DNA fragment containing a GRE-like sequence was incubated with in vitro-translated GR protein and collected on streptavidin beads. The DNA fragments with mutated GRE-like sequences were also used to ascertain the binding specificity of GR. DNA-bound GR was detected by Western blot with anti-GR antibody.