Ab139178

The primary antibodies used in this study were as follows: MitoProfile Total OXPHOS Rodent WB Antibody Cocktail [NADH dehydrogenase (ubiquinone) 1β subcomplex 8 (NDUFB8), succinate dehydrogenase complex subunit B (SDHB), ubiquinol-cytochrome c reductase core protein II (UQCRC2), ATP synthase, H+ transporting, mitochondrial F1 complex, α-subunit (ATP5A), ab110413, Abcam], NADH dehydrogenase (ubiquinone) iron-sulfur protein 4 (NDUFS4; ab139178, Abcam), cytochrome c oxidase subunit IV (COX IV; ab14744, Abcam), and CS (ab129095; Abcam). The following secondary antibodies were used in the present study: rabbit anti-goat IgG (H&L) (#A102PT; American Qualex, San Clemente, CA, United States ) and mouse anti-goat IgG (H&L) (#A106PU; American Qualex).

Protein lysates were isolated from LV tissue samples as described previously [21 (link), 22 (link)]. Independent biological replicates for each group were run separately on SDS-PAGE gels and transferred to nitrocellulose membranes using standard procedures. Primary antibodies against Auh (ab155980, Abcam), Crat (ab153699, Abcam), Decr1 (sc-366484, Santa Cruz), Hadha (sc-82185, Santa Cruz), Ndufs4 (ab139178, Abcam), Tim23 (611222, BD; control for mitochondrial structural integrity), Tom40 (sc-365466, Santa Cruz; control for mitochondrial structural integrity), and tubulin (T9026, Sigma-Aldrich; loading control) were used. For immunodetection, secondary antibody donkey anti-rabbit, anti-goat, or anti-mouse (Dianova) and ECL™ Prime Western Blotting Reagent (Amersham) were used. Data were quantified with the ImageJ 1.41 version software (http://rsbweb.nih.gov/ij/).

Proteins separated by SDS-polyacrylamide gel electrophoresis in 8% or 12% gels were transferred to polyvinylidene difluoride (PVDF) membranes that were blocked with PVDF blocking reagent for Can Get Signal (Toyobo, Osaka, Japan) for 1 h then incubated with primary antibodies against SHMT2 (1:1,000; #12762; Cell Signaling Technology, Danvers, MA, USA), NDUFS4 (1:1,000; ab139178; Abcam, Cambridge, UK), NDUFA9 (1:1,000; ab14713; Abcam), MT-CO1 (1:1,000; ab14705; Abcam), SDHA (1:1,000; #11998S; Cell Signaling Technology), or β-ACTIN (1:5,000; A1978; Sigma-Aldrich, St. Louis, MO, USA) over night at 4 °C [Can Get Signal immunoreaction enhancer solution 1 (Toyobo) was used for dilution]. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies against rabbit IgG (1:10,000; G-21234; Thermo Fisher Scientific, Waltham, MA, USA) or mouse IgG (1:10,000; G-21040; Life Technologies/Thermo Fisher Scientific, Carlsbad, CA, USA) for 1 h at room temperature [Can Get Signal immunoreaction enhancer solution 2 (Toyobo) was used for dilution]. Bands were detected with a bio-imaging analyser (EZ-Capture ST; ATTO, Tokyo, Japan) using ECL Select western blotting detection reagent (GE Healthcare, Little Chalfont, UK).