Ab33985

The 20 μg aliquots of cytoplasmic and mitochondrial fractions were mixed with SDS-loading buffer and boiled at 95 °C for 3–5 min. Equal amounts of proteins were loaded and electrophoretically separated using SDS-PAGE (Pulilai Co, Beijing, China). The separated proteins were transferred to nitrocellulose membranes and the membranes then incubated at 4 °C overnight with the following primary antibodies: anti-PINK1 (1:1000, ab23707, Abcam, Boston, MA, USA), anti-parkin (1:1000, ab77924, Abcam), anti-LC3B (1:1000, #3868, Cell Signaling Technology, Danvers, MA, USA), anti-P62 (1:1000, #39749, Cell Signaling Technology), anti-Tomm20 (1:1000, #42406, Cell Signaling Technology), anti-COX IV (1:1000, ab33985, Abcam), anti-Drp1 (1:1000, #5391, Cell Signaling Technology) anti-Mfn2 (1:1000, #9482, Cell Signaling Technology), anti-OPA1 (1:1000, #80471, Cell Signaling Technology), anti-cleaved caspase-3 (1:1000, #9661, Cell Signaling Technology) or anti-β-actin (1:5000, ab8226, Abcam). The membranes were then incubated with the appropriate secondary antibodies for 1 h at room temperature. The protein bands were visualized using enhanced chemiluminescence (Amersham Bioscience, Uppsala, Sweden) and quantified using Image J software (NIH).

After 48 h of ELIT treatment, cells were harvested as described previously by Torrens-Mas [35 (link)]. Protein content (supernatant) was determined with the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Ten micrograms of protein were resolved on a 12% SDS-PAGE gel and electrotransferred onto nitrocellulose membranes using the Trans-blot^® Turbo™ transfer system (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% non-fat powdered milk in TBS with 0.05% Tween for 1 h. Antisera against OXPHOS complexes (ab110411; Abcam, Bristol, UK), SOD-1 (574597, Calbiochem^®, San Diego, CA, USA), SOD-2 (sc-30080; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CAT (219010, Calbiochem^®, San Diego, CA, USA), GRd (sc-133245; Santa Cruz Biotechnology, Santa Cruz, CA, USA), 4-HNE (HNE11-S, Alpha Diagnostic, San Antonio, TX, USA), COXIV (ab33985, Abcam, Bristol, UK), PGC1α (ab54481, Abcam, Bristol, UK), and GAPDH (sc-25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as primary antibodies. Protein bands were visualized as described previously by [34 (link)].

Four-micron-thick renal paraffin sections were used for IHF staining. Briefly, after paraffin section dewaxing, rehydration, antigen repair, permeability, and blocking, the renal tissues were incubated with anti-F4/80 rabbit polyclonal antibody (Servicebio, GB11027, 1:500, Wuhan, China), anti-fibronectin (FN) rabbit polyclonal antibody (Servicebio, GB114057, 1:200, Wuhan, China), anti-α-SMA rabbit polyclonal antibody (Servicebio, GB111364, 1:400, Wuhan, China) and anti-NLRP3 rabbit polyclonal antibody (Servicebio, GB11300, 1:600, Wuhan, China) at 4°C overnight. After rewarming, the kidney tissue was incubated with a secondary antibody at room temperature for 1 hour. After staining the nucleus, the renal paraffin sections were observed and photographed under a fluorescence microscope. For costaining, after dewaxing, rehydration, antigen repair, permeability and blocking, anti-LC3B (Proteintech, 14600-1-AP, 1:200) antibody and anti-COXIV antibody (Abcam, ab33985, 1:500) were incubated in renal tissue simultaneously at 4°C overnight. After rewarming, the kidney tissue was incubated with anti-mouse and anti-rabbit secondary antibodies at room temperature for 1 hour simultaneously. Then the nuclei were stained and photographed.

L_4–6 spinal cord segments were rapidly collected and homogenized on ice due to their vulnerability to ischaemic injury [20 (link)]. The cytosolic and nuclear fractions were extracted and purified with a nuclear and cytosol protein extraction kit (KangChen, KC415, Shanghai, China) [18 (link)]. The mitochondrial fractionation was performed according to a multiple centrifugation method described previously [15 (link)]. Briefly, the homogenates were first centrifuged at 750 g at 4 °C and then at 8000 g for 20 min at 4 °C. The pellets obtained were considered the mitochondrial fraction. The samples were separated by gel electrophoresis and then transferred to polyvinylidene difluoride membranes. After blocking with 5% non-fat milk, the membranes were incubated overnight at 4 °C with primary antibodies against p53 (Abcam, ab26, 1:200), PUMA (Abcam, ab9643, 1:200), cleaved caspase 3 (Cell Signaling Technology, #9661, 1:300) and NF-κB p65 (phospho S536, Abcam, ab86299, 1:100). β-actin (Abcam, ab8227, 1:10000), histone (Abcam, ab10799, 1:5000) and COX-IV (Abcam, ab33985, 1:5000) were used as the controls. The bands were visualized by an enhanced chemiluminescence (ECL) kit (Beyotime, Beijing, China) and quantified using Quantity One software (Bio-Rad Laboratories, Milan, Italy).

At the time of tissue harvest, heart was excised and rinsed in phosphate‐buffered saline to remove excess blood on tissue and in ventricles. Hearts were then submerged into OTC compound and frozen in liquid nitrogen. For hearts with I/R injury, sections were collected 500 μm below suture line to capture the injured region. Sections were stained with hematoxylin and eosin for evaluation of general cardiac morphology and tissue organization. Masson's trichrome staining was used to visualize cardiac fibrosis. Fibrosis was quantified from low power microscope images by dividing the arc length of fibrotic scar to the circumference of left ventricle.
For confocal microscopy, frozen sections were fixed in cold acetone. Non‐specific antigen binding was blocked by incubating sections with 5% donkey serum prior to incubating sections with antibody against ABCB8 (1:100) (Ardehali et al, 2005) and COX4 (1:100, ab33985, Abcam) in 4°C overnight. Species‐specific secondary antibody with different fluorophore (1:200, Jackson Immunochem) was used to visualize the antigen, and nucleus were counterstained with TO‐PRO‐3 stain (Life Technologies) according to manufacturer's instructions. Images were acquired on a Zeiss LSM510 confocal microscope.