Figure 7.

Exogenous expression of oATP8 protein does not negatively impact mitochondrial function

In gel activity analysis in 12-week-old non-transgenic and transgenic C57BL/6J(mtC57BL/6J) and C57BL/6J(mtFVB) mice to detect ATPase activity. Mouse liver mitochondria were purified and ∼25 μg mitochondrial protein was used for clear native-PAGE. ATPase activity was documented and stained with Coomassie to detect ATP synthase monomer and dimer formation (Ai and Aii). Quantitative assessment of ATP hydrolysis activity was performed using isolated liver mitochondria from 12-week-old male mice (B). The V_max and K_m of ATP synthase to ATP substrate was calculated with the 95% confidence interval (CI). Lines were fit using “Non-linear fit: Michaelis-Menten” in GraphPad Prism. Values are means ± SEM (n = 5 animals) (Table S1). ATP synthesis and maximal respiratory capacity were monitored in isolated liver and skeletal muscle mitochondria from 9- to 12-week-old male mice. Error bars show SEM. Two-way ANOVA was performed with no significant differences between groups (n = 5 for liver mitochondria, n = 3 for skeletal muscle mitochondria) (D and E). The sensitivity of the ATP synthase to oligomycin was assessed by monitoring ATP hydrolysis activity. Values are means ± SEM (n = 3 animals) (C). Lines were fit using “Non-linear fit: [Inhibitor] vs. normalized response – Variable slope” in GraphPad Prism and IC₅₀ values and 95% CI were calculated (Table S2).