Mitochondrial Protein Acetylation Analysis

Isolated mitochondrial protein (20–30 µg) were loaded onto precast 4–12% Bis-Tris mini gels (Invitrogen, Waltham, MA) for gel electrophoresis and then transferred to a PVDF membrane. Acetylation of mitochondrial proteins was detected by anti-acetylated-lysine (Ac-K²-100) (1:1000, Cell Signaling Technology). For mitochondrial fractions of fetal heart ventricles, VDAC/Porin (1:1000)(Abcam, Cambridge MA) was used as the loading control [27 (link)]. Density values of each of the sample bands were normalized to their corresponding loading control as relative expression. A peroxidase-conjugated secondary antibody (SeraCare Life Sciences, Gaithersburg, MD) was used for all immunoblots.