Ab57602

The primary antibodies used in the present study were as follows [30 (link)]: Drp1 (1:1000, Abcam, #ab56788), Fis1 (1:1000, Abcam, #ab71498), Opa1 (1:1000, Abcam, #ab42364), Mfn1 (1:1000, Abcam, #ab57602), Mfn2 (1:1000, Abcam, #ab56889), Mff (1:1000, Cell Signaling Technology, #86668), Bcl2 (1:1000, Cell Signaling Technology, #3498), Bax (1:1000, Cell Signaling Technology, #2772), caspase9 (1:1000, Cell Signaling Technology, #9504), survivin (1:1000, Cell Signaling Technology, #2808), p53 (1:1000, Cell Signaling Technology, #9282), complex III subunit core (CIII-core2, 1:1000, Invitrogen, #459220), complex II (CII-30, 1:1000, Abcam, #ab110410), complex IV subunit II (CIV-II, 1:1000, Abcam, #ab110268), Tom20 (1:1,000, Abcam, #ab186735) [31 (link)].

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.).

Protein expression was analyzed as previously described^{25 (link)}. Briefly, frozen human left ventricles (LV) in each group were homogenized in an ice-cold lysis buffer containing protease and phosphatase inhibitors (5872 S; Cell Signaling), centrifuged and the supernatant was transferred to a new tube. Total protein concentration was determined using a BCA protein assay kit according to the manufacturer’s instructions (23225; Thermo Scientific). Lysates were loaded and separated using SDS-PAGE and then transferred onto a PVDF membrane. The following antibodies were used in the present study: anti- phosphorylated (S616) (1:1000; 3455; Cell Signaling) and total Dynamin-like protein 1 (DRP1) (1:1000; ab56788; Abcam) anti- Mitofusin 1 (MFN1) (1:2000; ab57602; Abcam), anti-Mitofusin 2 (MFN2) (1:2000; ab56889; Abcam), anti- Optic Atrophy 1 (OPA1) (1:1000; 612606; DB Biosciences), total OXPHOS human WB antibody cocktail (1:2000; ab110411; Abcam), anti-ATP5A subunit (1:1000; sc-136178; Santa Cruz) and anti-GAPDH (1:1000; ab8245; Abcam).

Cells were homogenized in lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 % Triton X-100, protease inhibitor cocktail) and incubated for 15 min on ice. Protein lysates (25 μg) were resolved on 4–20 % mini protean TGX Gel (Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membrane. Immunoblots were performed with anti-MFN1 (1:1000, #ab57602), p62 (1:1000, #ab91526), p84 (1:500, #ab487) and PGC1-α (1:200, #ab77210 (Abcam, Cambridge, UK), FIS1 (1:1000, #ALX-210-1037, Enzo Life Sciences, New York, NY, USA), Tubulin (1:4000, #T6199) and TOMM20 (1;500, #WH0009804M1) (Sigma Aldrich), CASP9 (1:500, #9502), CASP3 (1:1000, #9662) and LC3 (1:1000, #2775) (Cell Signaling, Danvers, MA, USA), and TDP-43 (1:500, #10782-2-AP, Proteintech, Chicago, IL, USA) antibodies. The Clarity Western ECL Substrate (Bio-Rad) was used for chemiluminescence detection. Densitometric analyses were performed using QuantityOne software (Bio-Rad).

Primary antibodies used for Western Blotting were anti-GAPDH (MAB374; Millipore), VDAC1 (ab14734; Abcam), MFN1 (ab57602; Abcam), MFN2 (ab56889; Abcam), OPA1 (612607; BD Biosciences), FIS1 (10956-1-AP; Proteintech), MFF (17090-1-AP; Proteintech), DRP1 (Dlp1; 611112; BD Biosciences), involucrin (I9018; Sigma), profilaggrin (sc66192; Santa Cruz), HSP70 (ADI-SPA-810; Enzo life science), alpha-tubulin (sc8035; Santa Cruz). The following secondary antibodies were used: horseradish peroxidase-conjugated rabbit anti-mouse (P0161; DAKO) and horseradish peroxidase-conjugated goat anti-rabbit (PO448; DAKO). Hepes, D-Mannitol, Sucrose, Ethylenediaminetetraacetic acid (EDTA), Trizma Hydrochloride (Tris-HCL), Sodium chloride (NaCl), Igepal (NP-40), Sodium deoxycholate (DOC), Sodium dodecyl sulfate (SDS), Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were purchased from Sigma Aldrich.

Cardiac tissue samples were prepared in lysis buffer containing 50 mm Tris–HCl, 150 mm NaCl, 1 mm EGTA, 1 mm EDTA, 1% Triton X-100, and protease inhibitor mixture (Roche Applied Science) and run on Invitrogen NuPAGE Bis–Tris gels. The protein expression levels in the cardiac tissue were analyzed using standard western blotting techniques with the following antibodies for Mfn1 (Ab57602, Abcam, 1:1000 fold dilution), Mfn2 (9482S, Cell Signalling, 1:1000 fold dilution), Cx43 (3512S, Cell Signalling, 1:6000 fold dilution), phospho-Cx43 (48-3000, Invitrogen, 1:1000 fold dilution), GAPDH (60004-1-1g, Proteintech, 1:5000 fold dilution).