Vdac1

A 20 mg piece of frozen mixed ventricle was homogenized in 125 μL of sucrose buffer for subsequent extraction of the mitochondrial, nuclear, and cytosolic fractions [39 (link)]. Proteins were assayed as previously described [40 (link)] and incubated for 16 h with primary antibodies (diluted 1:100 to 1:4000) against GCLC, GCLM (provided by Dr. Forman), Nox4, Vdac1 (Abcam, Cambridge, MA, USA), NF-κB p65, acetylated-NF-κB (Lys 310), IκBα, p-IκBα, H3, (Cell Signaling, Danvers, MA, USA), Keap1, Nrf2, GSK3β, Parkin, GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 4-hydroxy-2-nonenal (4HNE), Nox2 (EMD Millipore, Burlington, MA, USA), and Hmox1 (Proteintech, Rosemont, IL, USA). Blots were visualized using an Odyssey system (LI-COR Biosciences) and quantified using ImageJ. Nuclear and cytosolic extractions were tested for purity against H3 and GAPDH [39 (link)]. Mitochondrial extractions were also tested for purity with Vdac1 and GAPDH. In addition to consistently loading the same amount of total protein per well, values were further normalized by correcting with the densitometry values of Ponceau S staining [41 (link)]. Three Western blots were run per protein to include all available biological replicates. All blots contained no less than 2 representative samples per group.

The methods used for protein extraction from in vitro and in vivo samples and for western blot analysis have been described previously (Yu et al., 2017 (link)). The antibodies used include BNP (Abcam, ab236101, 1:500), MHC (Abcam, ab180779, 1:1000), Vinculin (Abcam, ab129002, 1:500), p62 (Cell Signaling Technology, #5114, 1:1000), LC3 (Abcam, ab48394, 1:500), β-actin-HRP (Kang Cheng, KC-5A08, 1:5000), VDAC1 (Abcam, ab154856, 1:1000), Ubiquitin (PTM BIO, PTM-1107, 1:2000), PINK1 (Abcam, ab23707, 1:500), Parkin (Abcam, ab77924, 1:500), FUNDC1 (Abcam, ab224722, 1:500), PHB2 (Santa Cruz, sc133094, 1:1000), BNIP3L (Cell Signaling Technology, #12396, 1:1000), p-Parkin (Ser65) (Affinity Biotech, AF3500, 1:1000).

The following antibodies were used: Actin (Sigma-Aldrich, St. Louis, MO, USA, #A4700 1:2500), Actin (Sigma-Aldrich, #A5441 1:2500), LC3 (Cell Signaling Technology, Danvers, MA, USA #3868 1:1000), Mfn2 (Abcam, Cambridge, UK, #Ab56889 1:1000), Neuropeptide Y (Cell Signaling Technology, #11976T 1:500), pSer293-PDH (Merck Millipore, Burlington, MA, USA #ABS204 1:1000), PDH (Santa Cruz, Dallas, TX, USA #sc-377,092), SNAP25 (Biolegend, San Diego, CA, USA #850301 1:1000), Synapsin-1 (Cell Signaling Technology, #5297T 1:1000), Synaptophysin (Abcam,#Ab14692 1:1000), Synaptotagmin-1 (Novus Biologicals, Littleton, CO, USA #MAB4364-SP 1:500), Syntaxin-1 (Sigma-Aldrich, #S0664) 1:500), TIM23 (BD Biosciences, Franklin Lakes, NJ, USA #611223 1:1000), and VDAC1 (Abcam, #Ab14734 1:1000).

Cells were lysed in ice-cold RIPA buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) in the presence of Protease Inhibitor Cocktail (Roche) for 10 min. Lysates were clarified by centrifugation and incubated with 2 μg of antibodies for 16 h followed by Protein A/G agarose for 1 h at 4°C. The immunocomplexes were washed 3 times with cold RIPA buffer and resuspended in 15 μl of 2 × SDS sample buffer for SDS-PAGE. Commercial antibodies used in this study were: FLAG-tag mAb (Sigma-Aldrich, F3165), c-myc tag (BETHYL, A190-205A), GAPDH (GeneTex, GTX100118), MDA5 (Enzo, ALX-210-935), RIG-I (AdipoGen, AG-20B-0009), 14-3-3 Family Antibody Sampler Kit (Cell Signaling Technology, #9769), VDAC1 (Abcam, ab15895), ACSL4/FACL4 (OriGENE, TA324720), MAVS/IPS-1 (Enzo, ALX-210-929-C100), Tubulin (Cell Signaling Technology, #2128).

Heart lysates were prepared in RIPA buffer containing protease and phosphatase inhibitors. Heart lysate or cytoplasm and mitochondria fractions were loaded into 4–12% Bis-Tris NuPAGE gels (ThermoFisher) or 4–15% Mini Protean gels (Bio-Rad). Proteins were transferred to nitrocellulose membrane using Turbo-blotter (Bio-Rad). Primary antibodies [CaMKII (Abcam), 1:1000; phospho-CaMKII (Thermo Scientific), 1:1000; VDAC1 (Abcam), 1:2000; FLAG (Rockland), 1:2000; GAPDH (Cell Signaling), 1:10,000; CKmito (Sigma), 1:6000; CoxIV (Cell Signaling), 1:1000; CK-M (Sigma), 1:10,000; α-actinin (Sigma), 1:1000; OxPhos (Abcam), 1:500; AcLys (Cell Signaling), 1:1000; HA (Sigma), 1:5000; SERCA2a (Badrilla), 1:5000; PDH (Abcam), 1:1000] were incubated with the membrane overnight at 4 °C. Secondary antibodies were incubated with the membrane at room temperature for 1 h. Blots were imaged using an Odyssey Fc Imager (Licor). Bands were quantified using Image Studio Software (Licor), coomassie-stained membranes were quantified using Image J software.