Fig. 1. COX-2 and iNOS bind selectively in vitro and in intact cells. (A) RAW264.7 cells were treated with LPS (2 µg/ml) and IFN- (100 U/ml). COX-2 was immunoprecipitated by COX-2–specific antibody and analyzed by Western blot with antibodies against COX-2 and iNOS. Control indicates untreated cells. (B and C) RAW264.7 cells were treated with LPS–IFN- with or without an iNOS inhibitor 1400W (100 µM) or COX-2 inhibitor SC58125 (100 µM). Cell lysates were subjected to immunoprecipitation (IP) and Western blot analysis with antibodies against COX-2 and iNOS. (D) The fragments of iNOS denoted in red bind to full-length COX-2, whereas fragments labeled purple do not, as determined by coimmunoprecipitation of full-length COX-2 by iNOS fragment fused to glutathione S-transferase (GST). The numbers represent the number of the amino acid sequence. (E) Transfected HEK293T cells expressing COX-2 and iNOS fragments expressed as fusion proteins with GST were precipitated with glutathione-conjugated beads. Proteins were detected by Western blot with antibodies against GST or COX-2. (F) Transfected HEK293T cells expressing COX-2 and epitope-tagged (Myc) iNOS fragments were immunoprecipitated with Myc-specific antibody and then analyzed by Western blot. (mock: The cells were treated with transfection reagent without the plasmid.) (G) Generated fragments of COX-2 that bind to full-length iNOS are labeled in red; those that do not bind are labeled in yellow. (MBD, membrane-binding domain). (H) Transfected HEK293T cells expressing iNOS and Myc-tagged COX-2 fragments were immunoprecipitated with Myc-specific antibody and analyzed by Western blot. (mock: The cells were treated with transfection reagent without the plasmid.) [View Larger Version of this Image (94K JPEG file)]

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