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.