Anti sdhb

For immunohistochemical staining, after antigen retrieval, the slides were blocked with a 5% BSA solution for 1 ​hour and incubated with mouse anti-CD68 (1:100, Invitrogen, USA), anti-iNOS (1:100, Abcam, UK), anti-CD163 (1:1000, Proteintech, USA), anti-TNFα (1:100, ZenBio, China), anti-IL1β (1:100, Proteintech, USA), anti-IL10 (1:100, Proteintech, USA), anti-TGFβ1 (1:100, Proteintech, USA), anti-MMP9 (1:100, Proteintech, USA), anti-OCN (1:100, Affinity, China), anti-HIF1α (1:100, Proteintech, USA) and anti-SDHB (1:100, Proteintech, USA) at 4 ​°C overnight. Goat anti-rabbit IgG (1:100, Servicebio, China) was used as a secondary antibody and the nuclei were stained using 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Servicebio, China). Images were captured using Aperio AT2 (Leica, German).

Total protein was obtained using RIPA lysis buffer. Cell membrane, cytoplasm and mitochondrial proteins were obtained using the cell membrane protein and cytoplasmic protein extraction kit (Beyotime) and the cell mitochondrial isolation kit (Beyotime). BCA protein assay Kit (APPLYGEN) was used to quantitate the protein levels. The primary antibody information used is as follows: anti-CLDN10 (1:1000; Abcam, ab52234), anti-Flag (1:1000; CST, 14793S), anti-ATP5O (1:2000; Abcam, ab110276), anti-Acetyl-ATP5O (1:200; Abcam, ab214339), anti-SIRT3 (1:1000; Abcam, ab217319), anti-NDUFS2 (1:5000; Abcam, ab192022), anti-Cleaved-Caspase 3 (1:1000; Affinity, AF7022), anti-E-cadherin (1:10000; Abcam, ab40772), anti-N-cadherin (1:5000, Abcam, ab76011), anti-SDHB (1:5000, Proteintech, China), anti-GAPDH (1:8000, Proteintech, China), anti-ATP1A1 (1:8000, Proteintech, China) and anti-COX Ⅳ (1:8000, Proteintech, China).

Harvested cell pellets were resuspended in the mammalian protein extraction reagent (Thermo Fisher Scientific) containing protease inhibitor mixture (Roche Applied Science) and 5-mM sodium orthovanadate (Sigma), incubated on ice for 20 min, and vortexed for 10 min followed by centrifugation for 25 min at 20,000g at 4 °C. The protein concentration was determined using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Proteins were resolved using a 12.5% (% T) SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked with 7% (w/v) skim milk and probed with antibodies: anti-Sdha (1:2000; Cell Signaling Technology), anti-SDHB (1:4000; Proteintech), anti-Pkm (1:10,000; Cell Signaling Technology), anti-pPkm (1: 10,000; Cell Signaling Technology), anti-Shpk (Carkl) (1:2500; MyBioSource), and anti-β-actin (1: 25,000; Fitzgerald). Data from three independent experiments were used in densitometric analysis, whereas only a representative Western blot is shown for brevity.