Anti mt co1

Protein extracts were separated by SDS-PAGE (10% gels) at room temperature at 80 mV and then transferred to 0.2 µm-pore nitrocellulose membranes at 4 °C at a total of 600 mA using a Mini-PROTEAN Tetra Cell and a PowerPac Basic, both from Bio-Rad. Membranes were blocked with 5% non-fat milk 0.05% Tween 20 TBS for 1 h at room temperature, then incubated with primary antibodies overnight at 4 °C. Antibody dilutions were: anti-MTCO1 (Abcam ab90668) 1:1 000; anti-SDHA (Abcam ab137040) 1:6 000; anti-β-tubulin (Sigma-Aldrich T0198) 1:5 000. After washing in 0.05% Tween TBS, blots were incubated for 2 h with anti-mouse or anti-rabbit peroxidase-conjugated secondary antibodies (Calbiochem, San Diego, CA, USA) at dilution of 1:5 000. Protein bands were detected using EZ-ECL reagents (Biological Industries) and scanned with Dyversity 4 (Syngene, India). UN-SCAN-IT (Silk Scientific, Inc., USA) was used for densitometry analysis.

For immunoblotting, cells were chilled on ice, rinsed with ice-cold PBS twice and lysed with RIPA buffer (Sigma-Aldrich, R0278) plus the protease inhibitor cocktail (Thermo Scientific, A32963). Protein concentrations were determined using the BCA Protein Assay Kit (BioRad). About 25 μg protein per sample was loaded on a 10% SDS-PAGE gel and transferred to the PVDF membrane, which was washed with PBS Tween 20 buffer (PBST) (ThermoFisher Scientific, #28360) for 5 minutes three times, blocked with 5% milk in PBST, and incubated with the anti-DAP3 (Abcam, #ab11928), anti-MT-CO1(Abcam,# ab14705), anti-MT-ND1(Abcam,# ab181848), anti-β-actin (Cell signaling technology,# 2146S), anti-DR5 (Cell signaling technology,#8074T), anti-cleaved caspase I (Cell signaling technology,# 2225T), anti-GSDMD (Abcam,# ab219800), anti-cleaved caspase 3 (Cell signaling technology, # 9661S), or the antibody directed against denatured core protein in the cold room overnight. The anti-core antibody was prepared in our lab using the recombinant core protein [33 (link)]. After washing with PBST for 10 minutes three times, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibody (Abcam) at room temperature for one hour. The membrane was washed with PBST for 10 minutes three more times and subjected to chemiluminescent analysis. All experiments were repeated at least three times.

Mitochondrial and cytoplasmic fractions, which were previously boiled in the Laemmli sample buffer, were analyzed by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE), were transferred on nitrocellulose membrane and blotted against various antibodies. We used anti-GFP (rabbit polyclonal, EnzyQuest, Vassilika Vouton, Greece), anti-MTCO1 (mouse monoclonal, abcam, Cambridge, UK) and anti-α-tubulin (mouse monoclonal, Develomental Studies Hybridoma Bank, Iowa City, IA, USA) antibodies.