Figure 3.

Induction of iNOS and NO synthesis in HUVECs cultured in low-Mg conditions. HUVECs were cultured in 0.1 or 1.0 mM Mg-containing medium for 24 h. All the experiments were performed in triplicate at least three times. NO levels in culture medium were measured by the Griess method (a) and mass spectrometry (b). (c) Western blot was performed to evaluate the total amounts of iNOS, eNOS, and eNOS/p-Ser¹¹⁷⁷ (p-eNOS). Anti-GAPDH antibodies were used as a control of equal loading. A representative blot and densitometry performed on three independent experiments and obtained by ImageJ are shown. (d) The mRNA expressions of iNOS were analyzed by real-time PCR. (e) NO levels were measured in the medium of the cells using the Griess method after silencing iNOS. Non-silencing scrambled sequences (NS) were used as controls. * p ≤ 0.05; ** p ≤ 0.01; and *** p ≤ 0.001.