CVEC were silenced for bovine eNOS and transfected with the phospho-mimetic or the dephospho-mimetic form of human eNOS in Thr495 (A) or Ser114 (B) (T495D and T495A or S114D and S114A, respectively). To verify cell transfection, western blot for eNOS Thr495 (A, upper panels) or Ser114 phosphorylation (B, upper panels) was carried out and normalized for total eNOS In these cells cGMP levels were: (pg/mg protein): 3.5 ± 0.4 for S114D and 3.3 ± 0.2 for T495D, p<0.01 vs. Vector 7.2 ± 0.3; 7.5 ± 0.2 for S114A and 6.9 ± 0.4 for T495A) of and ROS/RNS levels were (peak area/mg protein): 96 ± 4 for S114D and 98 ± 2 for T495D, p<0.01 vs. Vector 135 ± 3; 129 ± 4 for S114A and 132 ± 4 for T495A). The graphs represent quantification of phospho-Akt/total Akt gels (Arbitrary Densitometric Units); ***p<0.001 vs. Vector and ##p<0.01 vs. T495D or S114D respectively. C. Akt activity in CVEC exposed to L-NAME (15 min, 200 µM). The graph represents quantification of gels (Arbitrary Densitometric Units).**p<0.01 vs. 0.1% BCS D Cleaved caspase-3 in CVEC exposed to L-NAME (6 h, 200 µM) in 0.1% BCS. The graph represents quantification of gels (percent ± S.D.). ***p<0.001 vs. 0.1% BCS. E Nitrated-Akt in CVEC treated with L-NAME (15 min, 200 µM) in 0.1% BCS. The graph represents quantification of gels (Arbitrary Densitometric Units). ***p<0.001 vs. 0.1% BCS. In the presence of L-NAME the number of cells was: 153 ± 6, 200 µM L-NAME, vs. 0.1% BCS 105 ± 4; p<0.01.