Cox 4 antibody

Precipitated protein samples from the population study were processed and analysed by SDS-PAGE and western blotting as described elsewhere56 (link). Membranes were probed with Phospho-p38 MAPK antibody (Thr180/Tyr182, clone: 12F8) and COX IV antibody (loading control), both from Cell Signaling. Peroxidase-conjugated Donkey Anti-Rabbit IgG (H +L) antibody (Jackson ImmunoResearch) was used as secondary antibody and visualized by the use of Supersignal West Pico (Pierce Technology, Inc., IL, USA). Chemiluminescence was detected using an Imagequant LAS4000 imager and the protein bands were quantitated with the use of Imagequant TL Software (both from GE Healthcare Life Sciences).

DHTDK1 antibody (Proteintech, 1:1000), β-Actin antibody (Proteintech, 1:5000), Total OXPHOS antibody (Abcam, 1:1000, this OXPHOS antibody contains five mouse mAbs, one each against Complex I NDUFB8, Complex II SDHB, Complex III UQCRC2, Complex IV MTCO1, and Complex V ATP5A), COX IV antibody (Cell Signaling, 1:1000), anti-phospho AKT (Cell Signaling, Ser473, 1:1000), anti-phospho AKT (Cell Signaling, Thr 308,1:1000), anti-AKT (Cell Signaling, 1:1000), anti-phospho ERK1/2 (Cell Signaling, 1:2000), anti-ERK1/2 (Cell Signaling, 1:1000), anti-phospho p38 (Cell Signaling, 1:2000), anti-p38 (Cell Signaling, 1:1000), anti-phospho JNK (Cell Signaling, 1:1000) and anti-JNK (Cell Signaling, 1:1000) were diluted in 5% milk, Tris-buffered saline TBS, and 0.1% Tween 20. Secondary antibodies (Sigma, Anti-Rabbit IgG, A3687; Sigma, Anti-Mouse IgG, A3562) were then used at 1:20000 dilution. Total protein was quantified prior to loading, and equal amounts of protein (30µg) loaded per lane. Bands were imaged by fluorescence, and relative protein abundance estimated using ImageJ.

Diaphragm muscles were prepared in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS) with protease and phosphatase inhibitor cocktails (Sigma). The protein concentration was determined using a Coomassie Plus protein assay reagent (Thermo Scientific). For each sample, 10 µg of protein were loaded and separated on a SDS-polyacrylamide gel, according manufactures direction (BioRad). The proteins were then transferred to Nitrocelluloseous membranes blocked with milk and incubated with Rabbit polyclonal anti-peptide antibody that can recognize myonectin (epitope 77-KQSDKGI NSKRRSKARR-93). Myonectin antibody was kindly provided by the lab of GW Wong and had been used previously (Seldin et al., 2012 (link)). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was purchased from Novus Biologicals (NB300-221), COX IV antibody was purchased from Cell Signaling Technology (4844), and FNDC5 antibody was purchased from Abcam (ab131390). Antibody detection was performed with the appropriate horseradish perioxidase-conjugated secondary antibodies. Visualizations were completed with MultiImage III FuorChem^® M (Alpha Innotech) and quantifications were performed by Alphaview Software (Alpha Innotech).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) and ROS scavenger N-acetyl-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hochest 33342, carboxy-H2DCFDA, MitoProbe DiOC2 (3) Assay Kit and MitoProbe Transition Pore Assay Kit (M34153) were obtained from Invitrogen (USA). Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) Apoptosis Detection Kit was purchased from Keygen (Nanjing, Jiangsu, CHINA). RPMI1640 medium and fetal bovine serum (FBS) were from Hyclon (Logan, UT, USA). Caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Z-LEHD-FMK and caspase-family inhibitor Z-VAD-FMK were from Calbiochem (Gibbstown, NJ, USA) and Beyotime (CHINA). Antibodies against caspase-3, caspase-4, caspase-8, caspase-9 and COXIV antibody were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against cytochrome c, Bax, Bcl-2, p38, p-p38 and anti-goat LgG-HRP were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-β-actin and anti-GAPDH primary antibodywere from Sigma-Aldrich (St. Louis, MO, USA). Anti-rabbit LgG-peroxidase and anti-mouse IgG-peroxidase were from Vector (Burlingame, CA, USA).Mitochondria isolation kit was purchased from Pierce (Pierce, IL, USA), protein assay kit were purchased from Bio-Rad (Hercules, CA, USA), and the ECL detection kit were from Applygen Technologies Inc (Beijing, CHINA).

Icariin (United States, 489-32-7) and ferric ammonium citrate (FAC, United States, 1185-57-5) were purchased from Sigma-Aldrich (Supplementary Figure S1). Cleaved caspase-3 (Asp175) antibody (United States, #9661), MFN2 antibody (United States, #11925), DRP1 antibody (United States, #8570), COX IV antibody (United States, #4850), RUNX-2 antibody (United States, #12556), active β-catenin antibody (United States, #19807), cyclin D1 antibody (United States, #2922), PI3K antibody (United States, #4257), P-PI3K antibody (United States, #4228), AkT antibody (United States, #4691), P-AkT antibody (United States, #4060), mTOR antibody (United States, #2972), P-mTOR antibody (United States, #5536), P-ERK antibody (United States, #4370), ERK antibody (United States, #4695), P-p38 antibody (United States, #4511), p38 antibody (United States, #8690), P-JNK antibody (United States, #4668), JNK antibody (United States, #9252) were all purchased from Cell Signaling Technology. BAX antibody (United States, 50599-2-Ig), FIS1 antibody (United States, 10956-1-AP), cytochrome C antibody (United States, 66264-1-Ig) and osteopontin (OPN) antibody (United States, 22952-1-AP) were all purchased from Proteintech (United States, 50599-2-Ig). Bcl-2 antibody was purchased from R&D system (United States, MAB8272). Anti-beta actin antibody was purchased from BOSTER (China, BM3873).