Fig. 1.
COX-2 interacts with nNOS. (a) Western blot analysis of basal level COX-2 expression in cortex, hippocampus, hypothalamus, and cerebellum. (b) Colocalization of nNOS and COX-2 in cerebellar granular cell cultures. The localizations of COX-2 and nNOS were determined by immunohistochemistry. Cerebellar granular cells were fixed, permeabilized, blocked, and then incubated with COX-2 and nNOS antibodies. Immunostaining was visualized by confocal microscopy. Images of COX-2 (green) and nNOS (red) are superimposed to show colocalization. Nuclei were visualized with Hoechst staining (blue). (c) Coimmunoprecipitation of COX-2 and nNOS in mouse brain cerebellum after seizure. Mice were subjected to maximal electrical shock-induced seizures. Cerebellum lysates were immunoprecipitated (IP) with COX-2 antibody and analyzed with antibodies against mouse COX-2 and rabbit nNOS. (d) Coimmunoprecipitation of COX-2 and nNOS in HEK293T cells. Cell lysates were immunoprecipitated with COX-2 antibody and analyzed by Western blotting with antibodies against mouse COX-2 and rabbit nNOS. (e) Mapping COX-2 sites mediating binding to nNOS. HEK293T cells were transfected with Myc-COX-2 fragments and full-length nNOS. Cell lysates were immunoprecipitated with myc antibody and analyzed by Western blotting with anti-myc antibody and anti-nNOS antibody. (f) Mapping nNOS sites mediating binding to COX-2. HEK293T cells were transfected with full-length COX-2 and myc-nNOS fragments. Cell lysates were immunoprecipitated with COX-2 antibody and analyzed by Western blotting with anti-COX-2 antibody and anti-myc antibody. All experiments were repeated at least five times, and representative figures are shown.