Fig 1.
Validation of acetylated and succinylated lysine antibodies.
(A-B) Three commercial anti-acetylated lysine antibodies i.e. 05–515 clone 4G12 (Millipore), sc-8649 (Santa Cruz), #9441 (CST) were compared by three different methods, i.e. (A) Mitochondrial lysates extracted from different mouse tissues were subjected to western blotting. (B) In vitro non-enzymatic acetylation assay using acetyl-CoA or sodium acetate (NaOAc; negative control) was conducted followed by western blot analysis. (C) In vitro deacetylation assay using mouse liver mitochondria and eluted recombinant GST-SIRT3 or GST-SIRT3 bound to GSH-agarose was performed and subsequently subjected to western blotting. (D) Subcellular fractionation of mouse liver homogenate or C. elegans was performed to isolate nuclear fraction (nuclear), postmitochondrial supernatant (PMS) and mitochondria enriched fraction (mito) for western blotting. Absence of signal in the PMS and a similar pattern in the nuclear and mito fraction that corresponds to the abundance of mitochondrial NDUFS3 indicates mitochondria-specific succinylation.
Fig 2.
Mitochondrial acylation increases to a similar degree in carbonylation with ageing in C. elegans.
Subcellular fractionations of C. elegans homogenates were performed followed by western blotting using anti-acetylated lysine antibody(A) or anti-succinylated lysine antibody(B). Protein carbonyl content was determined using slot blot followed by derivaziation and detection using anti-2,4-dinitrophenylhydrazine antibody(C). (Ai, Bi) The western blots shown are representative of three independent experiments. Densitometry was then performed to quantify the mitochondrial acylation status. The data shown in (Aii, Bii, Cii) represent the average (±SD) of three independent experiments. (D) Oxygen consumption of Day 4 and Day 12 worms measured using Seahorse flux analyzer. Bar graphs shown represent basal respiration (i), maximal respiration (ii), spare respiration (iii) and non-mitochondrial respiration (iv) of C. elegans. (A-D) Statistical analysis was performed using unpaired t-test, * = p≤0.05, ** = p≤0.01, *** = p≤0.001 and **** = p≤0.0001.
Fig 3.
Mitochondrial succinylation increases with ageing in D. melanogaster.
D. melanogaster were homogenized and the whole fly lysates were used for western blotting using anti- succinylated lysine antibody. The bar graph represents the average (±SD) of three independent experiments. Statistical analysis was performed using unpaired t-test, * = p≤0.05and ** = p≤0.01.
Fig 4.
Mitochondrial acylation in young and old rats.
Subcellular fractionations of rat liver, heart and brain tissues were performed followed by western blotting using anti- acetylated lysine (A) or anti-succinylated lysine (B) antibody. Densitometry was then performed to quantify the mitochondrial acylation status. (C) Subcellular fractionations of rat, i.e. Wistar and SD brain tissues were performed followed by oxygen consumption measurement using a Clarke electrode and the state 3, state 4 and maximal respiratory rates are shown. The data shown in (A, B, C) represent the average (±S.E.M), expressed as fold increase compared to young rat. Each data point represents an individual rat.