Supplementary Material for: Endothelial Nitric Oxide Synthase Phosphorylation at Threonine 495 and Mitochondrial Reactive Oxygen Species Formation in Response to a High H<sub>2</sub>O<sub>2</sub> Concentration

<b><i>Background:</i></b> Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr<sup>495</sup>) in human umbilical vein endothelial cells (HUVECs) exposed to H<sub>2</sub>O<sub>2</sub>. <b><i>Methods:</i></b> HUVECs were exposed to 400 μM H<sub>2</sub>O<sub>2</sub> for 30 min. Phosphorylation at Thr<sup>495</sup> was assessed by Western blotting and reactive oxygen species (ROS) monitored by flow cytometry. Protein kinase C (PKC) pathways were investigated by pretreatment with PKC-β inhibitor ruboxistaurin or pan-PKC inhibitor GF109203X. In addition, we investigated ROCK and ERK pathways by MEKK1/2 inhibitor U0126 and ROCK inhibitor Y27632. <b><i>Results:</i></b> H<sub>2</sub>O<sub>2</sub> increased eNOS phosphorylation at Thr<sup>495</sup> (to 176% vs. control (100%), p < 0.001) along with increased mitochondrial ROS formation (from 19.7 to 45.3%, p < 0.01). This rise in phosphorylation could be prevented by U0126 and Y27632 in a dose-dependent manner, but did not result in lowered mitochondrial ROS formation. Conversely, addition of the antioxidant N-acetyl-L-cysteine only prevented mitochondrial ROS formation but did not prevent phosphorylation of eNOS Thr<sup>495</sup>. <b><i>Conclusion:</i></b> H<sub>2</sub>O<sub>2</sub>-mediated phosphorylation of eNOS Thr<sup>495</sup> is mediated by ROCK and ERK activity, but not by PKC, and is uncoupled from mitochondrial ROS signaling. Furthermore, ERK inhibition increased mitochondrial ROS formation.