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Published in final edited form as: Circulation. 2011 Jul 25;124(6):731–740. doi: 10.1161/CIRCULATIONAHA.111.030775

(A) HAECs were infected with LacZ or Nox4 adenovirus for 24h, the cells lysed, and assessed for phosphorylated (Ser-1177; p-eNOS) and total eNOS as well as actin by immunoblotting. *p<0.05 vs. Ctl by Student's t-test; N=3. (B) HAECs and RaSMCs were co-cultured and assessed for cGMP accumulation as described in Methods. The left panel represents basal activity, whereas the right panel demonstrates the cells after stimulation with the calcium ionophone, A23187 (10μM). *p<0.05 by Student's t-test; N=3. (C) HAECs were infected with Nox4 alone or Nox4 with catalase for 48h and assessed for total and phosphorylated (Ser-1177) eNOS, catalase, and actin by immunoblotting. (D) HAECs were cultured for 18h in hypoxic conditions +/- Ad-Nox4i and probed for total and phosphorylated (Ser-1177) eNOS, Nox4 and actin by immunoblotting. (E) Aorta were harvested from wild-type (WT) and VE-Cad Nox4 transgenic (TG) mice and assessed cGMP accumulation (*p < 0.05 vs. WT by Student's t-test; n = 4). (F) Aorta were harvested from wild-type (WT) and VE-Cad Nox4 transgenic (TG) mice and assessed for contraction in response to phenylephrine (PE; *p<0.05 vs. WT by two-way repeated measures ANOVA, n = 7/group). (G) Aorta were harvested from eNOS-/- and VE-Cad Nox4 transgenic (TG) eNOS-/- mice and assessed for contraction in response to phenylephrine (n = 5/group).

(A) HAECs were infected with LacZ or Nox4 adenovirus for 24h, the cells lysed, and assessed for phosphorylated (Ser-1177; p-eNOS) and total eNOS as well as actin by immunoblotting. *p<0.05 vs. Ctl by Student's t-test; N=3. (B) HAECs and RaSMCs were co-cultured and assessed for cGMP accumulation as described in Methods. The left panel represents basal activity, whereas the right panel demonstrates the cells after stimulation with the calcium ionophone, A23187 (10μM). *p<0.05 by Student's t-test; N=3. (C) HAECs were infected with Nox4 alone or Nox4 with catalase for 48h and assessed for total and phosphorylated (Ser-1177) eNOS, catalase, and actin by immunoblotting. (D) HAECs were cultured for 18h in hypoxic conditions +/- Ad-Nox4i and probed for total and phosphorylated (Ser-1177) eNOS, Nox4 and actin by immunoblotting. (E) Aorta were harvested from wild-type (WT) and VE-Cad Nox4 transgenic (TG) mice and assessed cGMP accumulation (*p < 0.05 vs. WT by Student's t-test; n = 4). (F) Aorta were harvested from wild-type (WT) and VE-Cad Nox4 transgenic (TG) mice and assessed for contraction in response to phenylephrine (PE; *p<0.05 vs. WT by two-way repeated measures ANOVA, n = 7/group). (G) Aorta were harvested from eNOS-/- and VE-Cad Nox4 transgenic (TG) eNOS-/- mice and assessed for contraction in response to phenylephrine (n = 5/group).