Anti uqcrc2

Two-dimensional blue native PAGE was performed as described previously^{67 (link)}. Briefly, skeletal muscle, which was collected from one mouse per each of the four groups, was homogenized with a glass Teflon homogenizer in buffer containing 10 mM HEPES-KOH (pH 7.4), 0.22 M mannitol, 0.07 M sucrose, and 0.1 mM EDTA. The extracts were then centrifuged at 500 g and the mitochondrial fraction was precipitated by further centrifugation at 10,000 g. The mitochondria (100 μg mitochondrial protein) were suspended in 10 μl of a buffer containing 50 mM Bis-Tris and 1 M 6-aminocaproic acid. Digitonin was added to solubilize the mitochondria. After a 30-min incubation at 4 °C, the solubilized proteins were obtained from the supernatant fraction by centrifugation at 22,000 g. The solubilized proteins were supplemented with 1 μl of sample buffer (5% Coomassie brilliant blue G-250 in 0.5 M 6-aminocaproic acid). A stacking gel (4%) and separating gels with a stepwise gradient of 8, 9, 10 and 11% were cast and electrophoresed. The second-dimension SDS-PAGE and immunoblotting were performed according to standard protocols, and the blot was probed with anti-NDUFA9 (#459100), anti-succinate dehydrogenase complex, subunit A (SDHA) (#459200) (Invitrogen, Waltham, MA), anti-UQCRC2 (#ab14745), and anti-RISP antibodies (#ab14746) (Abcam). Signal intensities of the blots were evaluated by ImageJ.

Total proteins and mitochondrial membrane proteins were isolated and separated as previously described [21 ]. Proteins were probed using anti-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated (p)-ERK (Thr202/Tyr204; Cell Signaling Technology), anti-p38 (Cell Signaling Technology), anti-phospho-p38 (Thr389; Cell Signaling Technology), anti-c-Jun N-terminal protein kinase (JNK; Cell Signaling Technology), anti-phospho-JNK (Cell Signaling Technology), anti-GRIM19 (Abcam, Cambridge, UK), anti-SDHA (Abcam), anti-UQCRC2 (Abcam), anti-MT-COI (Abcam), and anti-ATP-5A (Abcam) antibodies. Probed proteins were incubated with anti-rabbit (Cell Signaling Technology) or anti-mouse IgG (Cell Signaling Technology) secondary antibodies. Blot signals were detected using Super Signal West Pico Chemiluminescent substrate (Thermo Fisher Scientific).

Proteins were isolated from stably transfected cells at 90% confluence using the RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, China). Their concentrations were quantified using a BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China), and the proteins (30 µg/lane) were separated via SDS-PAGE before transfer to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Following this, the membrane with the proteins was subjected to the following incubations and washes: 5% lipid-free milk solution at 37°C for 1.5 h, primary antibody at 4°C overnight (ON), HRP-conjugated secondary antibody (1:1,000; Abcam, Shanghai, China) for 2 h at 37°C, and three TBST buffer washes. Finally, the protein signals were visualized using an enhanced chemiluminescence detection system with a chemiluminescent HRP substrate (Millipore, MA, USA). The antibodies used for protein detection were as follows: anti-UQCRC2 (1:1000, Abcam, Shanghai, China), anti-GAPDH (1:5000; Cell Signaling Technology, MA, USA), anti-cleaved caspase-3 (1:200, Cell Signaling Technology, MA, USA), anti-Bcl-2 (1:1000, Abcam, Shanghai, China), anti-Bax (1:1000, Abcam, Shanghai, China), and anti-Bak (1:10,000, Abcam, Shanghai, China).

Cells were collected and lysed with NP40 Lysis Buffer (Beyotime) with 1 mM Phenylmethanesulfonyl fluoride (Beyotime). Each quantity of 10⁶ cells was lysed with 50 μL NP40 buffer. The cell lysates were collected by centrifugation at 13,000 rpm at 4°C for 10 minutes. Protein loading buffer (Beyotime) was added, and the samples boiled at 95°C for 10 minutes. The isolated protein was subjected to SDS-PAGE on 4% to 20% polyacrylamide gels (ACE Biotechnology, Nanjing, China) and transferred to NC membranes (Millipore, St. Louis, MO, USA). The membranes were incubated with a primary antibody and secondary antibody. The primary antibodies included anti-Glut1, anti-LDHA, anti-NRF1, anti-NRF2, anti-GZMB, anti-Actin (Beyotime), anti-SIRT1 (Abclonal, Wuhan, China), anti-HK2, anti-CPT1α, anti-PGC1, anti-MTCO2, anti-TFAM, anti-GZMA, anti-GZMK, anti-PRF1, anti-PD-1, anti-TIM-3, anti-CTLA-4, anti-NDUFB8, anti-SDHB, anti-MTCO1, anti-UQCRC2, and anti-ATP5A (Abcam, Cambridge, UK). The secondary antibodies included HRP-labeled goat anti-rabbit and HRP-labeled goat anti-mouse antibodies (Beyotime). Finally, the proteins were detected by West Femto Maximum Sensitivity Substrate (ThermoFisher).

Liver tissues were homogenized with a glass-teflon homogenizer in a buffer containing 10 mM HEPES-KOH (pH 7.4), 0.22 M mannitol, 0.07 M sucrose and 0.1 mM EDTA as described^{3 (link)}. The liver extracts were centrifuged at 500 g and the supernatants were further centrifuged at 10,000 g to precipitate mitochondrial fraction. The mitochondrial fraction (100 μg protein) was suspended in 15 μL of a buffer containing 30 mM HEPES-KOH (pH 7.4), 150 mM potassium acetate and 10% glycerol. Digitonin (digitonin/protein ratio 8 g/g) was added to solubilize the mitochondria. After 30-min incubation at 4 °C, solubilized proteins were obtained as supernatant fraction by centrifugation at 22,000 g. Solubilized proteins were supplemented with 0.5 μL of sample buffer (5% coomassie brilliant blue G-250 in 1 M 6-aminocapronic acid). Stacking (4%) and separating gels with stepwise 8, 9, 10 and 11% were cast and electrophoresed according to the method of Scägger and von Jagow^{23 (link)}. Second-dimensional SDS-PAGE and immunoblotting were performed according to standard protocols, and blotted membranes were probed with anti-NDUFA9 (Invitrogen), anti-UQCRC2 (Abcam), anti-RISP (Abcam) and anti-COX1 (Abcam).