Research Paper Volume 15, Issue 23 pp 13608—13627

Figure 6. AGE improves endothelial cell functions in OxLDL-treated HUVECs. The indicated concentrations of AGE was treated in Ox-LDL-treated HUVECs for 48 h. (A) Immunoblotting was performed with anti-SIRT1, p-AMPK, AMPK, and β-actin antibodies in HUVECs. (B) Sirtuin activity and (C) NAD+ and NADH levels were analyzed, and NAD+/NADH ratios were quantified. (D) eNOS phosphorylation at Ser1177 and expression of total eNOS and β-actin in HUVECs were determined using immunoblotting. (E) eNOS was immunoprecipitated from HUVEC lysate, and immunoblotting was performed with anti-acetyl-lysine, SIRT1 and eNOS antibodies. (F) Immunoblotting was performed with anti-IRE1α SO3H and IRE-1α antibodies. (G) Heatmap depicting mRNA expression of the genes identified as RIDD substrates in HUVECs. (H) Ten μM GKT, 5 mM 4PBA, 1 mM NAC, or 1 mM AICAR were pre-treated in Ox-LDL-treated cells for 48 h. Immunoblotting was performed with anti-p-eNOS, eNOS, SIRT1, p-AMPK, AMPK, and β-actin antibodies and (I) sirtuin activity was measured, as described in Materials and Methods. ^#p < 0.05 compared to the control group; ^*p < 0.05 compared to the Ox-LDL only treated group. ^&p < 0.05 compared to the each counter AICAR not treated group. Values are presented as mean ± SEM (n = 3). Abbreviation: AGE: Angelica gigas NAKAI extract.