Ab154856

Liver tissue proteins were obtained from tissue lysates for western blotting. Protein concentration was determined using a BCA assay kit (Sigma-Aldrich, Saint Louis, MO, USA). Denatured proteins were separated on 12–20% Bis-Tris MOPS gels by SDS-PAGE and transferred to PVDF membranes. The PVDF membranes were incubated overnight at 4 °C with anti-Phospho-SAPK/JNK (Thr183/Tyr185) (1:1000, #4668, CST, Boston, MA, USA), anti-JNK (1:1000, #9252, CST, Boston, MA, USA), anti-VDAC1 (1:2000, ab154856, Abcam, Cambridge, UK), anti-Phospho-Src Family (Tyr416) (1:1000, #6943, CST, Boston, MA, USA), anti-Src (1:1000, #2123, CST, Boston, MA, USA), anti-Phospho-ATM (Ser1981) (1:400, sc-47739, Santa Cruz BioTech, Santa. Cruz, CA, USA), anti-ATM (1:4000, A1106, Sigma-Aldrich, MO, USA), anti-γH2AX (1:5000, ab81299, Abcam, Cambridge, UK), anti-H2AX (1:1000, 10856-1-AP, Proteintech, Wuhan, China), anti-p21(1:1000, ab188224, Abcam, Cambridge, UK) and anti-GAPDH (1:10,000, MB001, Bioworld Technology, Nanjing, China). The membrane-bound antibodies were detected by a hypersensitive chemiluminescence detection reagent from Fude Biological Technology Co., Ltd. (Hangzhou, China).

After treatment with indicated inhibitors of the complexes, the primary hepatocytes were washed twice with ice‐cold PBS and lysed with RIPA (Beyotime) supplemented with protease and phosphatase inhibitor cocktail (Roche). The lysates were boiled at 95°C for 10 minutes and separated by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE). Then, the separated proteins were transferred onto a nitrocellulose membrane (GE Healthcare). After blocked with 5% skim milk in the Tris‐Buffered Saline Tween‐20 (TBST) buffer for 1 hour at room temperature, the membrane was probed overnight at 4°C with primary antibody as listed below: phospho‐AMPKα (Thr172) (Cell Signaling Technology 2535), AMPKα (Cell Signaling Technology 5832) and β‐actin (Cell Signaling Technology 3700). Next day, the membrane was washed and re‐blotted with HRP‐conjugated secondary antibody (Cell Signaling Technology) at room temperature for 1h. Chemiluminescent HRP substrate (Millipore) was used to visualize protein bands by electrochemoluminescence (ImageQuant LAS4000). ImageJ was used to quantify the Western signals. Similar procedure was performed on frozen isolated liver mitochondria to detect mitochondrial complex I‐V expression using total OXPHOS rodent WB cocktail (Abcam, ab110413) and VDAC1 (Abcam, ab154856) primary antibody.

A polyclonal antibody against ASPH was raised in our institution using the synthetic peptide antigen of 12 amino-acid residues around the Fe²⁺-binding domain of ASPH. The antibodies against mitochondrial biomarkers heat shock protein 60 (ab46798) and voltage-dependent anion channel (ab154856), endoplasmic reticulum biomarkers calnexin (ab195198), green fluorescent protein (ab6556), H2AX (ab11175), POLG (ab207558) and mtTFA (ab176558) were purchased from Abcam (Cambridge, UK). The mouse monoclonal anti-myc (9E10) and HA antibodies (12CA5) were purchased from Sigma-Aldrich (St Louis, MO, USA) and Roche (Basel, Switzerland), respectively. Additional information on secondary antibodies is detailed in the Supplementary Information.

Proteins were separated on 13% SDS-PAGE gel, transferred to nitrocellulose membranes, and probed with various antibodies, including goat anti-SOD1 (C-17; SCB), sheep anti-SOD1 (Calbiochem), monoclonal anti-VDAC/porin 31HL (Calbiochem), goat anti-MIF (N-18, SCB), rabbit anti-MIF (FL-115, SCB), rabbit anti-human SOD1 (ab52950, Abcam), and rabbit anti-VDAC (ab154856, Abcam). Horseradish peroxidase-conjugated anti-mouse, anti-rabbit, anti-sheep, or anti-goat IgG secondary antibodies (Jackson Immunochemicals) were used and detected by ECL (GE Biosciences).

Mitochondrial-enriched fractions from the muscle (P2 and P3) were separated by denaturing NuPAGE 4%–12% Bis-Tris gels and blotted with anti-ATP6 (Abcam ab219825) and anti-VDAC1/porin (Abcam, ab154856) antibodies.