Immunoblotting of Mitochondrial Proteins from Mouse Brain

Synaptic mitochondrial lysates were prepared as described above from mouse brain tissue. Immunoblotting was completed as previously described [58 (link)]. Briefly, equal amounts of protein (10 μg) were loaded onto 4-12% Bis-Tris gels, transferred to nitrocellulose membranes, blocked, and incubated with the following antibodies overnight at 4°C: the oxidative phosphorylation (OXPHOS) panel (1:5000) (MS604; Mitoscences), SOD2 (1:10,000) (ab16956; Abcam), VDAC1 (1:10,000) (4661; Cell Signaling), ATP5H (1:10,000) (MS504; Mitosciences), MFN1 (1:2,000) (ab57602; Abcam), TFAM (1:4,000) (LS-C30495; LifeSpan Biosciences), DRP1 (1:2000) (D8H5, Cell Signaling), and SQSTM1 (1:8000) (PM045, MBL). The OXPHOS antibody panel is a mix of antibodies that include: NDUFB8, SDHB, UQCRC2, MTCO1, and ATP5A1. Since commonly used mitochondrial protein loading controls (VDAC1 and GAPDH) change during aging according to our proteomics and under different conditions [59 (link), 60 (link)], we performed Coomassie staining to confirm equal protein loading (Supplemental Figure 1). Ponceau staining was done to confirm equal protein loading for each membrane. Chemiluminescent bands were visualized with an Image Station 4000MM Pro and analyzed using Carestream Molecular Imaging software (both from Carestream Health, Inc.).