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. 2008 Jul 23;105(30):10537–10540. doi: 10.1073/pnas.0804852105

Fig. 2.

Fig. 2.

nNOS-derived NO stimulates COX-2 activity in vivo and in vitro by S-nitrosylation. (a) S-nitrosylation of COX-2 in HEK293T cells with or without NOS inhibitor l-NAME, determined by the biotin switch assay. HEK293T cells were transfected with nNOS and/or COX-2 in the presence and absence of 1 mM l-NAME. S-nitrosylated proteins were precipitated and analyzed with Western blotting using COX-2 antibody. (b) S-nitrosylation of constitutive COX-2 in brain cerebellum. Homogenates from wild-type and nNOS-knockout mice were subjected to the biotin switch assay and Western blot analysis. (c) NMDA and ionomycin stimulate the S-nitrosylation of COX-2 in cerebellar granular neurons. Cerebellar granular neurons were treated with either 300 μM NMDA or 1 μM ionomycin. S-nitrosylated COX-2 was determined by biotin switch assay and Western blotting with COX-2 antibody. (d) Dominant-negative peptide (nNOS-PDZ domain) blocks nNOS–COX-2 binding and S-nitrosylation of COX-2. HEK293 cells stably expressing nNOS were transfected with COX-2 alone or with combinations of COX-2 and different amounts of myc-nNOS-PDZ plasmids. After transfection, cell lysates were immunoprecipitated with nNOS antibody, and bound proteins were detected with COX-2 or myc antibodies. Quantification of immunoprecipitated blot is shown. S-nitrosylation of COX-2 was determined by biotin switch assay and Western blotting with COX-2 antibody. (e) NMDA activates COX-2 activity in the presence and absence of 1 μM cycloheximide in cerebellar granular neurons. COX-2 activity was assayed in the cell lysates of primary cerebellar granular neurons treated with 300 μM NMDA and 5 μM glycine for 5 min. Bars represent the mean ± SEM of three independent cell cultures performed in triplicate. Asterisks indicate statistically significant differences by Student's t test (*, P < 0.05). (f) Inhibition of nNOS and COX-2 prevents NMDA neurotoxicity. Cell viability was assessed by MTT assay. All experiments were repeated at least five times, and representative figures are shown. Bars represent the mean ± SEM of three independent cell cultures performed in triplicate. Asterisks indicate statistically significant differences by Student's t test (*, P < 0.05).