Anti traf3

Mitochondrial membrane potential assay kit (C3601), Fast silver staining kit (P0017S), and cell mitochondria isolation kit (C2006) were purchased from Beyotime (Shanghai, China). Reactive oxygen species (ROS) assay kit (KGT010-1) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (KGA312) were purchased from KeyGen Biotech (Nanjing, China). Duolink In Situ Probemaker (DUO92010/DUO92009) and Duolink flowPLA detection kit-Red (DUO94001) were purchased from Sigma-Aldrich (Saint Louis, USA). All siRNA and shRNA were purchased from Origene (Beijing, China). Anti-OPA1 [DF8587, 1:500 for Western blot (WB), 1/100 for immunofluorescence (IF), and 1:200 for immunoprecipitation (IP)], anti-TRAF3 (DF7181, 1:1000 for WB, 1:100 for IF, and 1:200 for IP), anti-MFN1 (DF7543, 1:1000 for WB, 1:100 for IF, and 1:200 for IP), anti-TLR4 (AF7017, 1:1000 for WB, 1:100 for IF, and 1:200 for IP) monoclonal antibodies and goat anti-rabbit/mouse-horseradish peroxidase-conjugated secondary antibody (S0001/S0002, 1:5000 dilution) were purchased from Affinity (Cincinnati, USA).

Western blot was performed as previously reported^{44 (link)}. Briefly, the membranes were incubated overnight at 4 °C with the following corresponding primary antibodies: anti-HECTD1 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HECTD2, anti-HECTD3 (1:800, BIOS, Beijing, China), anti-TRAF3 (1:200, Affinity Biosciences, Shanghai, China), anti-phospho-IKKβ (Tyr199), anti-phospho-IκBα (Ser32/Ser36), anti-phospho-p65 (Ser536) (1:500, Affinity Biosciences, Shanghai, China), anti-IKKβ, anti-IκBα, anti-p65 (1:1000, Proteintech, Wuhan, China), anti-GST tag, anti-His tag, anti-FLAG tag, anti-HA tag (1:5000, Proteintech, Wuhan, China), and anti-β-actin (1:3000, Proteintech, Wuhan, China) antibodies.