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Fig. 1. Wild-type and mutant NOS2-GFP chimeras. (A) The N-terminus of NOS2 was mutated by PCR, creating C3S, Myr, Myr/C3S and A2C mutant chimeras fused to GFP. We created both full-length NOS2 chimeras (residues 1-1144) and short chimeras (residues 1-94). The different NOS2-GFP constructs were inserted into a pCDNA3 vector that was used to transfect COS7 cells and the enzymatic activity (B), incorporation of radioactive palmitate and myristate (C) and protein expression (D) were determined. Enzymatic activity of NOS2 was determined using the Griess method as previously reported (Navarro-Lérida et al., 2004). Transfected COS7 cells were starved for 1 hour in DMEM without serum and were then metabolically labelled for 4 hours with either [³H]myristic acid (Myr) or [³H]palmitic acid (Palm). Cell lysate proteins were separated by SDS-PAGE and expression of the NOS2 chimeras was determined by western blotting with antibodies to GFF.

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