Anti cox 4

Capsaicin (Cap, purity > 98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Sirtinol, the specific inhibitor of SIRT1, was purchased from Selleckchem (S2804). Anti-SIRT1 antibody was purchased from Abcam (ab32441, USA). Anti-caspase-3, anti-Bcl2, anti-cytochrome c, and anti-Cox4 were purchased from Cell Signaling Technology (Beverly, MA). Anti-β-actin was purchased from the Jiancheng Bioengineering Institute of Nanjing. The horseradish peroxidase-labeled IgG secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA).

Tissues were collected and lysed in RIPA buffer (Sigma, St. Louis, MO, USA) in the presence of protease and phosphatase inhibitors (Halt™ protease and phosphatase inhibitor, Thermo Scientific, Waltham, MA, USA), and samples were prepared in protein sample buffer (0.25% bromophenol blue, 0.5M dithiothreitol, 50% glycerol, 10% sodium dodecyl sulfate (SDS), 0.25M Tris-Cl pH 6.8, and trace amounts of bromophenol blue), boiled, and stored at −80 °C. For gel electrophoresis and Western blot analyses, samples were run on 4–15% precast Tris-HCl SDS-polyacrylamide gels (BioRad, Hercules, CA, USA) and transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Blots were successively probed with anti-COX4 (Cell signaling technology, #11967S), -CRIF1 (Santa Cruz, #sc-374122), -caspase 8 (Cell signaling technology, #4790S), -caspase 9 (Cell signaling technology, #9508S), -BCL-2 (Cell signaling technology, #2876S), or -β-actin (Cell signaling technology, #4967S) antibodies at 1:1000 dilutions in TBS containing 3% protease free BSA (Sigma, St. Louis, MO, USA). Blots were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA) and the images were acquired and quantitated using Azure 300 Chemiluminescent Western Blot Imaging System (Azure Biosystems, Dublin, CA, USA).

For Western blotting, skeletal muscle tissues were homogenized with RIPA buffer. After the lysates were centrifuged at 13,000 rpm for 30 min, the supernatants were measured using bicinchoninic acid (BCA) (Thermo Fisher Scientific, Waltham, MA, USA) assay and boiled for 5 min at 100 °C after mixing with a 5× sample buffer. Western blotting samples were loaded onto SDS-PAGE gels, run, and transferred onto PVDF membranes (Merck Millipore, Burlington, MA, USA). After blocking with Tris Buffered Saline with Tween 20 (TBST) containing 5% skim milk, the membranes were incubated with primary antibodies overnight at 4 °C and then incubated with secondary antibodies for 1–2 h at RT. The membranes were incubated with ECL solution (Promega, Madison, WI, USA) and imaged using ImageQuant LAS 500 (GE Healthcare, Chicago, IL, USA).
The primary antibodies used were anti-BIS [1 (link)], anti-HSF1, anti-HSP70 (Enzo Life Sciences), anti-GAPDH, anti-HSPB5, anti-YAP1 (Santa Cruz Biotechnology), anti-cleaved PARP1, anti-desmin, anti-HSPB8, anti-p62 (ABCAm), anti-COX4 (Cell Signaling Technology, Danvers, MA, USA), and anti-filamin C (Novus Biologicals, Centennial, CO, USA).

RL71 (>97% purity, HPLC) was synthesized as described previously.^{14 (link)} anti-LC3B, anti-ubiqutin, anti-CHOP, anti-PARP, anti-p-JNK, anti-JNK, anti-p-PERK (Thr980), anti-p-AMPK (Thr172), anti-p-mTOR (Ser2448, 2971), anti-p-p70S6K (Thr389), anti-Cox4 and anti-cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-Grp78, anti-ATF4, anti-ATF6, anti-β-actin and anti-GFP antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-SQSTM1/p62, anti-ATG5 and anti-ATG7 antibodies were from Abcam (Cambridge, UK) and anti-LC3 was from Novus Biologicals (Littleton, CO, USA). GFP-LC3 plasmid was purchased from Yingrun Biotechnologies Inc. (Changsha, China). Compound C (CC) and BAPTA were obtained from Calbiochem (San Diego, CA, USA). zVAD was from Selleck Chemicals (Houston, TX, USA). Chloroquine (CQ), STO-609, SP600125 and PBA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ER-specific dye ER tracker Red, the mitochondrial specific dye MitoTraker Red CMXRos (M7512), JC-1 (T-3168), Fura-2/AM and Lipofectamine 3000 transfection reagent were purchased from Life Technologies (Grand Island, NY, USA). The Ca²⁺-ATPase activity assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing). All of the other chemicals were purchased from Sigma-Aldrich.

The detailed procedure has been described^{24 (link)}. The following primary antibodies were used: anti-Fxn (GeneTex, Cat.#: GTX54036), anti-Cox4 (Cell Signaling Technology, Cat.#: 48445), total OXPHOS Rodent WB Antibody Cocktail (MitoSciences, Cat.#: MS604), anti-Sod1 (Abcam, Cat.#: ab16831), anti-Sod2 (Enzo Life Sciences, Cat.#: ADI-SOD-111-D), anti-Sod3 (R&D Systems, Cat.#: AF4817), anti-Cat (Abcam, Cat.#: ab15834), anti-β-Tublin (Cell Signaling Technology, Cat.#: 2146 S), anti-Mmp9 (Proteintech, Cat.#: 10375-2-AP), anti-Col1a2 (Proteintech, Cat.#: 14695-1-AP), anti-Irp1 (Santa Cruz Biotechnology, Cat.#: sc-166022), anti-Tfrc (Abcam, Cat.#: ab84036), anti-Ft (Abcam, Cat.#: ab75973), anti-Opa1 (Novus Biological, Cat.#: NBP2-34206), anti-4-HNE (Abcam, Cat.#: ab48506) and anti-Gapdh (Cell Signaling Technology, Cat.#: 14C10). The proteins were detected and quantified using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).

Whole protein lysates were prepared using the PRO-PREP protein extraction solution (Intron Biotechnology, Seongnam, Korea), and mitochondrial and cytoplasmic fractions were performed with a mitochondria isolation kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of proteins were separated by electrophoresis on 8–12% SDS-PAGE gels and transferred onto nitrocellulose membranes (BD Biosciences, San Jose, NJ, USA). The membranes were blocked by incubation in blocking buffer (BD Biosciences) and probed with the following antibodies overnight at 4°C: anti-NeuN, anti-AT8, anti-Tau-5, anti-β-actin (Sigma–Adrich, St. Louis, MO, USA), anti-PSD95, anti-phospho(p)-Tau(T181), anti-p-Tau(S396; Abcam, MA, USA), anti-Drp1, anti-p-Drp1(S616), anti-COXIV, anti-GAPDH, anti-PARP, anti-cleaved caspase-3, p-Tau(S262), anti-CDK5, anti-ERK, anti-p-ERK, anti-GSK3β, anti-p-GSK3β(S9; Cell Signaling, MA, USA), and anti-p35 (Thermo Fisher Scientific, Waltham, MA, USA). The membranes were washed with TBS with 0.1% Tween-20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) for 1 h at room temperature. After washing with TBST, specific binding was detected using a chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA).