Fig. 4. Impairing mitochondrial function altered succinylation and protein distribution in the whole cell as well as in the mitochondria and non-mitochondrial fractions.

a The effects of rotenone (100 nM/20 min) on succinylation in HEK cells. After separation, mitochondrial and non-mitochondrial fractions were immune-precipitated with anti-succinyl lysine antibody and separated by SDS-PAGE followed by western blotting. The data from three different replicate experiments were expressed as the mean with error bars from standard error of the mean (SEM) (n = 3 independent experiments, two-way ANOVA followed by Bonferroni’s multiple comparisons test). b The effects of rotenone (100 nM or 5 μM/20 min) on the distribution of KGDHC protein between mitochondria and non-mitochondrial fractions. The data from three different replicate experiments were expressed as the mean with error bars from SEM (n = 3 independent experiments, two-way ANOVA followed by Tukey’s multiple comparisons test). c The effects of rotenone (100 nM, 5 μM/20 min) on the distribution of PDHC protein between mitochondria and non-mitochondrial fractions. The data from three different replicate experiments were expressed as the mean with error bars from SEM (n = 3 independent experiments, two-way ANOVA followed by Tukey’s multiple comparisons test). d Rotenone induces release of DLST into cytoplasm. In the control conditions, DLST (magenta) was concentrated inside mitochondria defined by COX-IV labeling (green). After 1 h of 100 nM Rotenone treatment, additional DLST labeling was found in the cytoplasm. Inserts on the right are magnified regions. Magenta: DLST; Green: COX-IV; Error bars represent SEM deviation from the mean (n = 98 fields from 19 dishes, two-way ANOVA followed by Bonferroni’s multiple comparisons test). Source data are provided as a Source Data file.