Research Paper Volume 15, Issue 23 pp 13608—13627

Figure 4. AGE regulates ER stress and IRE1α sulfonation in vascular dysfunction model. (A) Immunoblotting of p-IRE1α, IRE1α, sXBP-1, GRP78, CHOP and β-actin expressions in aorta and (B) respective quantitative analysis of protein expressions. (C) Immunoblotting with anti-IRE1α SO₃H and IRE1α antibodies in aortas. (D) Heatmap depicting mRNA expression of genes identified as RIDD substrates in the aorta. (E) RNA fold prediction in the secondary structure of mRNA fragments of sirt1. (F) In vitro cleavage assay using 1.5% denaturing agarose gel. Sirt1 mRNA cleaved by IRE1α with its two mutant mRNAs during the indicated times. (G) Immunoblotting of SIRT1, p-AMPK, AMPK, and β-actin in aorta from the indicated groups. (H) Sirtuin activity and (I) NAD⁺ and NADH levels were measured, and NAD⁺/NADH ratios were quantified. (J) Acetylated lysine levels and β-actin expression were determined using immunoblotting. (K) eNOS was immunoprecipitated from aorta tissues from the indicated groups, and its acetylated-lysine level was analyzed via immunoblotting with anti-acetyl-lysine and anti-SIRT1 antibody. Data are presented as mean ± SEM (n = 3, ^#p < 0.05 vs. NCD, ^*p < 0.05 vs. HFD). Abbreviations: NCD: normal chow diet; HFD: high-fat diet; AGE: Angelica gigas NAKAI extract.