Extended Data Fig. 3: Immunoblot validation, correlation analysis, and comparison of protein and NAD⁺ levels in aortic tissues categorized by degeneration and architecture scores.

a, Western blots of NAD⁺ metabolism-related proteins and differentiating markers of smooth muscle cells. b, Western blot using anti-acetylated lysine protein antibody in healthy (n = 6 patients) and degenerated (n = 7 patients) groups. SIRT3, NAMPT, NMNAT1, and SLC25A51 are proteins involved in NAD⁺ consumption, salvage, and mitochondrial transportation. TAGLN and MMP2 are markers of contractile status and synthetic status, respectively. Patient age and relative intensity of blots are marked below the blot stripes. Correlation between protein abundances of SLC25A51 and NAMPT (c), and SLC25A51 and NMNAT1 (d). NMNAT3 protein was not detected by LC-MS based characterization. Correlation between aortic architecture score and COL3A1 (e), and proline (f), quantified by LC-MS/MS. Pearson correlation coefficients and p-values determined by two-tailed Student’s t-tests are displayed. g, Log₂-transformed abundance of COL3A1 and proline in groups categorized by aortic degeneration (degenerated or healthy) quantified by LC-MS/MS. h, Log₂-transformed abundance of proteins associated with aortic disease. Log₂-transformed abundance of proteins associated with NAD⁺ metabolism with (i), or without (j), significant changes. k, Total levels of NAD⁺ and NADH (left), and the ratios of NAD⁺/NADH across patient subgroups: healthy (n = 10 patients), degenerated (n = 23 patients), degenerated with lower (n = 10 patients) or higher (n = 13 patients) aortic architecture score, measured by colorimetric assay. Related to Fig. 2. Data are represented as mean ± standard deviation, violin plots are presented as median and interquartile range, p-values were determined by Mann-Whitney U test or two-tailed Student’s t-tests.

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