Western and immunoprecipitation lysis buffer

Mitochondria in cultured cells were isolated according to previous study.^[52] Briefly, the collected cells were resuspended in hypotonic buffer which contained 10 × 10⁻³m Tris base, 10 × 10⁻³m NaCl, and 2.5 × 10⁻³m MgCl₂ at a pH of 7.5, homogenized on ice and centrifuged at 1300 g for 5 min at 4 °C. The supernatants were collected and centrifuged at 17 000 g for 15 min at 4 °C to precipitated the mitochondria, and the mitochondria was lysed with western and immunoprecipitation lysis buffer (Beyotime, Nanjing, China, Cat # P0013) for subsequent western blot assay. Liver mitochondria were isolated in terms of previous study.^[53] Briefly, liver tissues were washed with PBS followed by transferred in isolation buffer which contained 0.5 × 10⁻³m EDTA, 10 × 10⁻³m Tris, and 0.25 m sucrose at a pH of 7.4. The tissues were minced, washed and homogenized in 2.5 vol of isolation buffer. After increasing to an 8x initial vol with isolation buffer, the homogenates were centrifuged at 1000 g for 10 min at 4 °C and the supernatant fraction was centrifuged at 10 000 g for 10 min at 4 °C. The mitochondrial pellet was rinsed twice with isolation buffer, and the supernatant was collected as the cytosolic fraction. Freshly isolated mitochondria were used immediately for detection of mitochondrial complex activity.

Human cardiac myocytes were transfected with 1 μg of WT and variant plasmid DNA. Then, we harvested the cells 48 h after transfection. We used western and immunoprecipitation lysis buffer (Beyotime, Shanghai China) with protease and phosphatase inhibitor cocktail (Beyotime) to lysis cells. The proteins were subjected to 10% SDS/PAGE and were then transferred onto nitrocellulose membranes (Millipore, Burlington, MA, USA) and immunostained with rabbit anti‐NRDG4 antibody (dilution 1 : 500; Novus Biologicals, Centennial, CO, USA), rabbit anti‐GAPDH antibody (dilution 1 : 5000; Proteintech, Chicago, IL, USA) rabbit anti‐P27 antibody (dilution 1 : 1000; Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight. The membranes were incubated with HRP‐conjugated AffiniPure Goat Anti‐Rabbit IgG(H + L) secondary antibody (dilution 1 : 10 000; Proteintech) [21, 22]. All the raw immunoblots are available in the supplement (Figure S1).

R28 cells were lysed in Western and Immunoprecipitation Lysis Buffer (Beyotime Biotechnology, China) with Protease Inhibitor Cocktail (Abcam, UK). After incubated on ice for 5 min, the samples were collected and centrifuged at 12,000 rpm for 15 min. The total amount of protein was determined by Pierce™ BCA protein assay kit (Thermo Fisher Scientific, CA, USA). Proteins were electrophoresed on SurePAGE Gels (GenScrip, Shanghai, China) and transferred to PVDF membranes (Millipore, MA, USA). The membranes were incubated with anti-GTPBP3 (1:2000, Thermo Fisher Scientific, CA, USA) and anti-β-Actin (1:5000, Proteintech, Shanghai, China) overnight at 4 °C. After washing with TBST for three times, the membranes were incubated with secondary antibodies for 1 h at room temperature. The gray densities of GTPBP3 bands were normalized to the densities of β-actin bands with Image J software (National Institutes of Health, USA).

HT29, CT26, LOVO, MC38, and HCT116 cells were seeded in 96 well plate (5 × 10⁵ per well) overnight before CAP treatment. After 72 h of culture, cells were lysed using Western and immunoprecipitation lysis buffer 50 μL per well (Cat# P0013, Beyotime Biotechnology, Shanghai, China). The lysates were homogenized, and the resulting homogenates were centrifuged at 1,600xg at 4 °C for 10 min. The supernatants were collected and assessed using the Lipid Peroxidation Malondialdehyde (MDA) Assay Kit (Cat# S0131, Beyotime Biotechnology, Shanghai, China). Specifically, 200 µL of thiobarbituric acid (TBA) reagent was added to 100 µL of the cell suspension, and the mixture was heated in a boiling water bath for 15 min. After cooling and centrifuging the reaction mixture at 1,000xg for 10 min, the supernatant was separated, and absorbance was measured at 530 nm using the multi-mode microplate reader (SYNERGY H1, BioTek, Vermont, VT, USA). MDA levels were expressed as units (U) per 1 × 10⁶/mL cells and as nmol/mg protein.