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Fig. 5. Localisation of the sites of intracellular synthesis of ·NO in vivo, NOS2 activity in the presence of additional substrate and cofactor and effect of the NOS2 inhibitor 1400W and the ·NOS donor DETA-NONOate on the NOS2 fluorescence. (A) COS7 cells were transfected with the full-length wild-type NOS2-GFP and 36 hours post-transfection, the cells were incubated in vivo with 5 µM of the ·NO-sensitive probe diamino-Rhodamine-4M (DAR-4M) for 1 hour at 37°C. The location of the intracellular sites of ·NO synthesis is indicated in red. (B) COS7 cells transfected with the wild-type, C3S and Myr full-length NOS2-GFP chimeras were incubated with (shaded bars) or without (black bars) an excess of the NOS2 substrate L-Arg (10 mM) and the cofactor H₄B (10 µM) and ·NO-synthesising activity was determined. (C) Changes in the immunofluorescence signal of the wild-type full length NOS2-GFP chimera after addition of 100 µM of the NOS inhibitor 1400W or 100 µM of the ·NO donor DETA-NONOate. Arrows indicate the plasma membrane areas where the NOS2 GFP fluorescence can be observed. Cell nuclei were stained with Hoechst 33258 (blue).

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