Figure 7
Expression and phosphorylation of eNOS, expression of endothelin-1, and oxidative stress.
(A) mRNA expression of eNOS of HAECs cultured under normo- and hyperglycaemic conditions and after transfection with non-target (WT25mM+NT-siRNA) and GLO1 specific siRNA (WT25mM+GLO1-siRNA) (following 48 h siRNA transfection). (B–E) Western Blot analysis for eNOS (B), eNOS phosphorylation of Ser1177 (C), Ser615 (D), and Thr494 (E) of HAECs cultured under normo- and hyperglycaemic conditions and after transfection with non-target (WT25mM+NT-siRNA) and GLO1 specific siRNA (WT25mM+GLO1-siRNA) (following 72 h siRNA transfection). (N = 5). Different blots were used, with samples derived from the same experiment, analysed in the same concentration and in parallel to calculate the rate of phosphorylation of eNOS after determination of the concentration using β-actin as control on the same individual blot. lane 1: Precision Plus Protein Standard; lane 2, 6: WT25mM+GLO1-siRNA; lane 3, 7: WT25mM+NT-siRNA, lane 4, 8: WT25mM; lane 5, 9: WT5mM. (F) mRNA expression of endothelin-1 of HAECs cultured under normo- and hyperglycaemic conditions and after transfection with non-target (WT25mM+NT-siRNA) and GLO1 specific siRNA (WT25mM+GLO1-siRNA) (following 48 h siRNA transfection). (G) Endothelin-1 level were analysed extracellularly by ELISA. (N = 3, n = 3). ***p<0.001 vs. WT25mM+NT-siRNA, respectively. $$$p < 0.001 vs. WT25mM. (H) Mean fluorescence intensity of HAECs cultured under normo- and hyperglycaemic conditions and after transfection with non-target (WT25mM+NT-siRNA) and GLO1 specific siRNA (WT25mM+GLO1-siRNA) (following 72 h siRNA transfection). (N = 3, n = 3).
