Fig. 1.
The functional analysis of eNOS constructs. (A) Subcellular localization of eNOS in COS cells. eNOS constructs fused with RFP include: WT-eNOS-RFP, which is WT eNOS that primarily localizes at the Golgi apparatus, and RFP-eNOS-NLS to target the nucleus. (Scale bar: 10 μm.) (B) eNOS expression of eNOS constructs. (C) NO production in COS-7 cells transfected with eNOS constructs. Forty-eight hours after the transfection of eNOS constructs or control plasmid (RFP only), the media were processed for the measurement of nitrite, a stable breakdown product of NO in aqueous solution by chemiluminescence. (Left) NO release from samples collected from basal (4 h) accumulation of nitrite in serum-containing media is shown. The same cells were then incubated with serum-free DMEM for 6 h and the fresh media containing agonist ATP (100 μM) for 30 min. (Right) NO release as a result of ATP stimulation is shown. The cells were lysed to determine the total protein concentration and evaluate equal eNOS expression by Western blot. Data are presented as means ± SEM; n = 3; ∗, P < 0.01; ∗∗, P < 0.05.