Anti ndufa9

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.

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).

Protein lysates were prepared as previously described^{39 (link)} from differentiated ARPE-19 cells treated with or without 200 μM ddI in triplicate for 24 days. Total protein for each sample was quantified with a BCA kit and an equal amount of protein from each sample was separated by 4–15% gradient SDS-PAGE. Protein transfer and chemiluminescence detection were done as previously described^{40 (link)}. The primary antibodies used and dilutions were as follows: anti-AMPKα (Cell Signaling Technology, #2603), 1:1000, anti-pAMPKα (Cell Signaling Technology, #2535S), 1:1000, anti-NDUFA9 (Invitrogen, #459100), 1:500, anti-S6 (Cell Signaling Technology), 1:1000, anti-pS6 (Cell Signaling Technology), 1:1000, anti-γ-tubulin (Sigma Aldrich), 1:5000. The secondary antibodies used were HRP-conjugated anti-mouse and anti-rabbit (Jackson ImmunoResearch), 1:10000. Densitometry was performed with ImageJ (NIH).

Protein samples were lysed in 5 × SDS sample buffer containing beta-mercaptoethanol, boiled at 95°C for 10 min and loaded into each well for SDS-PAGE. Samples were then transferred to PVDF membrane (Thermo) and immunoblotted using anti-NDUFA9 (Invitrogen), anti-NDUFS3 (Invitrogen), anti-NDUFA13 (Invitrogen), anti-ATP5A (Invitrogen), anti-SDHA (CST), anti-VDAC (CST), anti-HSP60 (CST), anti-PHB1 (CST), anti-PDH (CST), anti-GAPDH (Sigma), anti-GRP78 (SCBT), anti-ABCA1 (SCBT), anti-FLOT1 (CST), anti-STAT3 (CST), anti-STAT1 (SCBT), anti-AMPK (CST), anti-ERK1/2 (CST), anti-p38 (CST), anti-SYP (Abcam), anti-ACSL4 (Abcam), all diluted in 5% BSA:TBST at 1:1000, followed by appropriate HRP-conjugated secondary antibodies (Thermo) incubation (diluted in 5% non-fat milk:TBST at 1:10,000), and developed using the SuperSignal^TM West Femto Maximum Sensitivity Substrate (Thermo).