Figure 5.

DHE inhibited transcriptional activation of COX-2 by effect on p300 recruitments and p50/p65 NF-κB nuclear translocation. The U87 cells were treated with DHE at the indicated concentrations for 48 h. A: The nuclear extracts were harvested for ChIP assay by using specific antibodies directed against p300, p65 and p50 to immunoprecipitate formaldehyde-fixed chromatin, followed by regular PCR with COX-2 primers. Normal IgG served as a negative control. B: The binding activities of p300, p65 and p50 to COX-2 promoter were analyzed by streptavidin-agrose pulldown assay. C: The total nuclear extracts were subjected to western blot analysis for p300, p65 and p50. The Lamin B1 used as a loading control. D: Laser scanning confocal microscope immunofluorescence assay was performed to detect the subcellular localization of p65, p50 and p300 and the co-localization of p50 with p65 or p300 by using specific antibodies against p65 (red), p50 (green) and p300 (red). And the typical morphology of cells was presented in the above.