Ab46798

Cells were grown to 70% confluence in DMEM with 10% FBS. Cells were lysed with 1× Passive Lysis Buffer (Promega) and ultrasonicated. Lysates were centrifuged (15,000 g, 10 min), and protein concentration in the supernatant was measured by the Micro BCA Protein Assay Kit (Thermo Fisher). Ten micrograms of total protein was resolved on 10% SDS–PAGE and blotted on nitrocellulose membranes, which were probed with specific antibodies. Amersham ECL Prime Western blotting detection reagent (RPN2232; GE Healthcare) was used as substrate for horseradish peroxidase (HRP). The specific antibodies used in this study were as follows: anti‐myc tag (ab9106, Abcam); anti HSP60 (ab46798, Abcam), anti‐ATP6 (MS508, MitoScience), and anti‐β‐tubulin (ab15568, Abcam); HRP‐conjugated goat anti‐rabbit (G‐21234, Thermo Fisher); and goat anti‐mouse antibodies (A10551, Thermo Fisher).

For proteins isolation, cells were lysed in RIPA buffer and quantified using Pierce BCA kit (Thermo-Fisher). For cytosolic/mitochondrial fractionation Cytochrome c Release Assay Kit (abcam, ab65311) was used. Proteins lysates (10–20 μg) were resolved on 5%-12% SDS–PAGE gels and transferred to PVDF membrane (Thermo-Fisher). Membranes were blocked in 5% Milk (BioRad) or 5% BSA (Sigma) in 1X TBST and incubated overnight at 4 °C in primary antibodies. Membranes were then washed with 1X TBST and incubated with secondary antibodies (Southern Biotech) for 1 h. The membranes were developed with ECL reagent (Thermo Fisher) on to X-ray films (Thermo-Fisher) using the chemiluminescence imager, AGFA CP100. Rabbit anti-HSPD1 (ab46798, 1:20,000) and rabbit anti-NLRC5 (ab117624, 1:1000) antibodies were purchased from Abcam; mouse anti-TOMM20 (H00009804-M01, 1:1000) was purchased from Abnova; anti-β-Actin (8H10D10) HRP conjugate (1:10,000) was purchased from Cell Signaling. Protein band density was quantified using ImageJ.

Each sample was loaded onto 4–20% Tris-glycine gels. After electrophoresis and transferring to nitrocellulose membranes, the membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 and 0.2% I-block (Tropix, T2015) for 90 min at room temperature. Membranes were then incubated overnight at 4 °C with following primary antibodies, anti-β-actin (1:1000, Sigma-aldrich A5441), anti-TOM40 (1:200, Santacruz, sc-11414), anti-ATP5A (1:500, Abcam, ab14748), anti-ACADM (1:500, Abcam, ab92461), and anti-HSP60 (1:500, Abcam, ab46798). After incubation with peroxidase-conjugated secondary antibodies, visualization was enhanced by chemiluminescence (GE Healthcare, NA931- anti-mouse, or NA934- anti-rabbit, or NA935- anti-rat). Optical density was assessed using the NIH Image analysis software.

Frozen cardiac tissues of mice were sectioned on the slide glass at a thickness of 10 μm. Slides were fixed with 4% paraformaldehyde or chilled methanol for 10 min. Heat shock protein 60 (ab46798, HSP60, Abcam) and poly (ADP-Ribose) (SM1398, PAR, ORIGENE, Rockville, MD, USA) antibodies were diluted at 1:100 and used to treat fixed samples for 24 h. Secondary antibodies tagged with FITC (for PAR, 1:200 dilution) and rhodamine (for HSP60, 1:200 dilution) were attached, and confocal images were obtained. Images were obtained using an LSM 700 Zeiss confocal microscope (Fluo-view, Carl Zeiss) at 488 nm (FITC), 530 nm (rhodamine), and 358 nm (DAPI for nucleus staining) and analyzed with ZEN software (ZEN 2009 light edition, Carl Zeiss). Images were collected from five separate preparations of cardiac tissue, and the results were averaged from all experiments.

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.

Cellular proteins were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was determined by the BCA method. A total of 40 μg of protein for each group was separated on 12% SDS-PAGE gels and transferred to 0.45-μm PVDF membranes (EMD Millipore). Membranes were then blocked with 5% BSA and incubated with primary antibodies at 4°C overnight against HPS70 (4876, CST), CD9 (ab92726, CST), CD63 (ab193349, Abcam), RBP4 (ab233138, Abcam), RPSA (ab133645, Abcam), RPS3(9538, CST), RPS20 (ab133776, Abcam), RPS14 (ab246916, Abcam), RPL4 (ab234829, Abcam), RPL13 (ab134961, Abcam), HSPD1 (ab46798, Abcam), HSPA8 (8444, CST), P-gp (13342, CST), p-cofilin-1 (3313, CST), cofilin-1 (5175, CST), PP1 (sc-7482, Santa Cruz), PP2A (9780, CST). Anti-β-actin (ab179467, Abcam) and anti-GAPDH (2118, CST) were used as the internal control. Anti-COX IV (11967, CST) was used as loading control for mitochondrial proteins. The membranes were then incubated at 37°C for 1 h with an HRP-conjugated secondary antibody (ab97051, Abcam). Bands were visualized by chemiluminescence according to the manufacturer’s protocols.

Immunoblotting was performed with anti-CPT1A (1/1,000) (ab128568; Abcam), anti-CPT1B (1/1,000) (ab134988; Abcam), anti-CPT1C (1/1,000) (ab87498; Abcam), anti-CTP2 (1/1,000) (ab181114; Abcam), anti-heat-shock protein-60 (HSP60) (1/1,000) (ab46798; Abcam), anti-TOMM20 (1/1,000) (ab56783; Abcam), anti-NDUFS1 (1/500) (sc-50132; Santa Cruz Biotechnology), anti-UQCRC2 (1/1,000) (ab14745; Abcam), anti-GFAP (1/500) (G6171; Sigma), anti-NDUFB8 (1/1,000) (ab110242; Abcam), anti-NDUFA9 (1/1,000) (ab14713; Abcam), anti-SDHA (1/1,000) (ab14715; Abcam), anti-MTCO1 (1/1,000) (ab14705; Abcam), anti-COX IV (1/1,000) (ab16056; Abcam), anti-Iba1 (1/1,000) (019-19741; Wako), anti-MAP2 (1/1,000) (ab32454; Abcam), anti-OLIG2 (1/1,000) (ab109186; Abcam), anti-PDHA1 (1/1,000) (no. 3205; Cell Signaling), anti-phosphoSer²⁹³-PDHA1 (1/1,000) (no. 31866; Cell Signalling), anti-β-Tubulin III (1/300) (T2200; Sigma) and anti-β-actin (1/30,000) (A5441; Sigma).