Fig. 4. COX-2–Myc fragment attenuates iNOS binding to COX-2– and NO-mediated activation of PGE₂ production. Transfected RAW264.7 cells expressing COX-2–Myc fragments 1 to 483 or 484 to 604 were treated with LPS–IFN-. (A) Cell lysates were immunoprecipitated with rabbit iNOS-specific antibody and analyzed by Western blot with antibodies against mouse iNOS, goat COX-2, and mouse Myc. (B) COX-2–Myc fragment (484 to 604) decreases S-nitrosylation of COX-2 in RAW264.7 cells. All the S-nitrosylated proteins were precipitated and COX-2 was detected by Western blot with COX-2–specific antibody. (C) Transfected RAW264.7 cells expressing the indicated COX-2 fragments were treated with LPS–IFN-. PGE₂ levels were measured and the data were pooled from three independent experiments performed, each in triplicate (*statistically significant by Student's t test). (D) PGE₂ and the indicated COX-2 fragments were visualized with confocal microscopy using antibodies against mouse Myc and rabbit PGE₂. Images of COX-2 (red) and PGE₂ (green) were superimposed to show colocalization. Nuclei were visualized with Hoechst staining (blue). In D1, arrows point to two RAW264.7 cells, only one of which is expressing the COX-2 fragment 484 to 604 (red). In D2, the same two cells are analyzed for presence of endogenous PGE₂ after activation of RAW264.7 cells by LPS–IFN- treatment. Immunofluorescent staining shows a reduction in the PGE₂ expression in cells expressing COX-2(484–604) compared with the nontransfected cell (D2). This observation contrasts with D4, where the arrows point to a nontransfected cell and a transfected cell expressing COX-2(1–483). D5 does not show a reduction of PGE₂ in the transfected cell as compared with the nontransfected cell. [View Larger Version of this Image (112K JPEG file)]

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