Anti atp5a1

Aliquots of cell lysate (50 µg/lane) were separated by electrophoresis on 10% or 12% SDS polyacrylamide gels. Anti-phospho-STAT6, anti-STAT6, anti-phospho-SMAD2 (#3104), anti-SMAD2, anti-phospho-SMAD3, anti-SMAD3, anti-phospho-SMAD1, 5, and 9, anti-SMAD1, 5, and 9, anti-phospho-ERK1/2 (44/42 MAPK) (#4370), anti-ERK1/2 (#9102), anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, and anti-SDHA (#5893) antibodies were purchased from Cell Signaling Technology (Beverley, MA, USA). Anti-NDUFA9 and COX-4 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-UQCRC2 (ab14745) was purchased from Abcam (Cambridge, UK). Anti-ATP5A1 was purchased from Invitrogen (Thermo Fisher). Secondary antibodies (goat anti-mouse and goat anti-rabbit) were obtained from Santa Cruz Biotechnology. Images were scanned on an ODYSSEY instrument and quantified using Image Studio Digits (LI-COR Biosciences, Lincoln, NE, USA). Full length uncropped blots are presented in Supplementary Figures 12–17.

After different treatments, the cells were incubated with RIPA lysis buffer containing a protease inhibitor cocktail on ice for 10 min. Cell lysates were then harvested by scraping, followed by brief sonication and centrifugation at 12,000 × g for 10 min at 4 °C. The protein concentration was determined via the BCA assay. Equal amounts of protein were separated by SDS/PAGE and the bands were electroblotted onto PVDF membranes. The membranes were blocked with 5% non-fat milk dissolved in TBST and then incubated with appropriate primary antibodies overnight at 4 °C. The following primary antibodies were used: anti-NLRP3 (1:1000, Abcam), anti-Gasdermin D-N-terminal (1:1000, Invitrogen), anti-caspase-1 (1:1000, Invitrogen), anti-cleaved caspase-1 (1:1000, Invitrogen), anti-GAPDH (1:10000, Abcam), α-Fodrin (1:1000, Cell Signaling Technology), anti-ATP5A1 (1:1000, Invitrogen), Calpastatin (1:500, ABclonal), and Flag (1:1000, Abcam). The membranes were then incubated with the following horseradish peroxidase-conjugated secondary antibodies: HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG purchased from Proteintech (1:5000, America). The immunofluorescent bands were then visualized and quantified using an ECL chemiluminescence kit (Absin, Shanghai, China) and a chemiluminescence–western blotting detection system (Tanon, Shanghai, China).

Oligodeoxynucleotides A*GTGTATTGCTTTGAGGAGGTAAGCTACAT* (Gln), A*ATGGGGTGATGTGAGCCCGTCTAAACATA* (Phe1) and A*AGTGTATTGCTTTGAGGAGGTAAGCTACATA* (Phe2) carrying C6-aminoallyl-modified nucleotides (marked by asterisks) were labeled with FluoroLink Cy3 monofunctional reactive dye (GE Healthcare) and were used as probes for in situ hybridization to mitochondrial tRNAs (http://www.einstein.yu.edu/labs/robert-singer/protocols/). Hybridization was carried out in 2× SSC containing 20% formamide. Mitochondria were detected with anti-ATP5A1 (1:100 dilution) (459240, Invitrogen). Slides were mounted in mounting media (90% glycerol, 1× PBS, 0.1 µg/mL DAPI, 1 mg/mL p-phenylenediamin). Raw images were acquired using NIS-Elements AR software on a Nikon TI-E/B inverse microscope with CFI Plan APO VC 100× 1.4 objective and a Hamamatsu OrcaR2 CCD camera (binning 1 or 2). Final images were prepared with Adobe Photoshop.