Tomm20

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).

Heart tissue was homogenized in a RIPA lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with protease inhibitor cocktail tablets (Roche Basel, Switzerland)) at 4 °C. After centrifuging for 30 min at 14,000× g, the supernatants were stored at −80 °C. For Western blot analysis, samples were boiled for 5 min in Laemmli sample buffer (5% SDS, 10% glycerol, 25 mM Tris-HCl pH: 6.8, 10 mM DTT, 0.01% bromophenol blue), and equal amounts of protein (30 μg) were separated on 8-15% SDS-polyacrylamide electrophoresis gels (SDS-PAGE). The relative amounts of each protein were determined with the following polyclonal or monoclonal antibodies: COX-1 (sc-1752 Santa Cruz Biotechnology, Dallas, TX, USA), COX-2 (160112Cayman Chemical Ann Arbor, Michigan USA); mitofusin 2 (ab56889Abcam Cambridge, UK,); TOMM20 (Sigma, WH0009804M1 Merck Life Science S.L.U. Darmstadt, Germany); β-actin (Sigma, A2066 Merck Life Science S.L.U. Darmstadt, Germany). After incubation with the corresponding horseradish peroxidase conjugated secondary antibody, blots were developed by the ECL protocol (GE Healthcare, Chalfont St Giles, UK). Protein band densities were normalized to β-actin. Densitometric analysis was carried out with Image J software and expressed in arbitrary units.

The following antibodies were used for immunoblotting: PARP (ThermoFisher, Cat# PA‐951; 1:1,000), TMBIM5 (Proteintech, Cat# 16296‐1‐AP; 1:2,000), VINCULIN (Cell signaling, Cat# 4650; 1:1,000), MCU (Sigma, Cat# HPA016480; 1:1,000), PHB1 (NeoMarkers, Cat# RB‐292; 1:500), AFG3L2 (Sigma, Cat# HPA004480; 1:5,000), ATPVa (Abcam, Cat# Ab14748; 1:5,000), TIMMDC1 (Abcam, Cat# Ab171978; 1:1,000), NDUFA9 (Invitrogen, Cat# 459100, 1:1,000), OMA1 (Santa Cruz, Cat# SC‐515788; 1:1,000), Total oxphos rodent (Abcam, Cat# Ab110413, 1:40,000), YME1L1 (Proteintech, Cat# 11510‐1‐AP; 1:2,000), Tubulin (Sigma, Cat# T6074; 1:5,000). The following antibodies were used for immunofluorescence: cytochrome c (ThermoFisher, 33‐8200; 1:500) and TOMM20 (Sigma, Cat# WH0009804M1; 1:500). Corresponding species‐specific HRP‐coupled antibodies were used for immunoblot (Biorad; Cat# 1706515 and Cat# 1706516) and fluorescently coupled secondary antibodies were used for immunofluorescence (Invitrogen Alexa Flour).

The following antibodies were purchased from the indicated suppliers: IRF1 (Santa Cruz; C20), IRG1 (ThermoFisher; PA5-102893 and Abcam ab222411), LAMP1 (Santa Cruz; H4A3), and TOMM20 (Sigma; HPA011562). Alexa Fluor 555-conjugated goat anti-rabbit and Hoechst 33342 dye were from Life Technologies. Ultrapure LPS (E. coli 0111:B4), TLR2 ligand FSL-1, and TLR8 ligand CL-75 were purchased from InvivoGen. Tocilizumab and infliximab were obtained from Roche and Pfizer, respectively. The JAK I inhibitor (CAS 457081-03-7) was obtained from Sigma. Itaconate and 4-octyl Itaconate were obtained from Sigma.