Fig 1: SIRT1 and SIRT3 deacetylate SOD3.Acetylated SOD3 was incubated with SIRT1 or SIRT3 and 2 mM NAD + for 30 min at 37 °C. Global deacetylation of SOD3 was evaluated by anti-acetylated-lysine immunoblotting (AI) and quantified by densitometry with normalization to total protein visualized with TCE labeling (AII). Data are expressed as mean with SD; a one-way ANOVA was performed on triplicates *p < 0.05, **p < 0.01. Site specific deacetylation was quantified by proteomics of trypsin digests in terms of fold change compared to acetylated SOD3 (B). ND indicates that acetylation at this site was not found in Ctrl SOD3.
Fig 2: Deacetylation of SOD3 restores furin cleavage.SOD3 acetylated with 1 mM sNHSAc and then deacetylated by SIRT1 or SIRT3 was incubated with furin overnight at 37 °C. All samples shown in (A) are incubated with furin, acetylation blocks furin cleavage as seen by a change in electrophoretic mobility in SDS-PAGE, and SIRT3 restores this shift (AI), the uncropped blot is included in Supp. Fig. 2B. SIRT3 has an observed MW of 33.5 kDa and can be seen as a band above cleaved SOD3. Anti-acetylated-lysine immunoblotting confirms acetylation and deacetylation of these samples (AII). The shift in SOD3 mass was also evaluated by intact mass spectrometry, integration of the deconvoluted spectra from 25700 to 27900 amu was performed and divided by the integration of 28075–28925 amu. This cleavage ratio for the AcSOD3 no-furin sample was subtracted from the cleavage ratio for each treatment and normalized to the SOD3 +furin sample. Percent cleavage is therefore relative to defining the SOD3 + furin cleavage ratio as 100 % or complete cleavage. SIRT1 restored 5.9 % of furin cleavage and SIRT3 restored 85.0 % (B).
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human Sirtuin 1/SIRT1 Protein, CF