siRNA duplexes targeting COX7RP, OGDH, DLST, DLD, MDH1, and MDH2 were synthesized using an algorithm that significantly improves the target specificity of siRNA, in particular, by efficiently estimating off-target sequences48 (link). A non-targeting control siRNA (siControl) used in this study was purchased from RNAi Inc. (Tokyo, Japan)48 (link). The siRNA sequences were detailed in Supplementary Table 9 . siRNAs were transfected into cells using RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cell lysates and mitochondrial fractions were prepared in a sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), heated at 100 °C for 15 min, and subjected to SDS-PAGE and western blotting analysis using antibodies for COX7RP (1:1000 dilution)2, RISP (1:5000 dilution, ab14746, Abcam, Cambridge, MA), COX1 (1:5000 dilution, ab14705, Abcam), COX4 (1:3000 dilution, 4844, Cell signaling, Danvers, MA), and β-actin (1:3000 dilution, AC-74, Sigma-Aldrich, St. Louis, MO).
Ab14746
Manufactured by Abcam
Sourced in United States
Ab14746 is a laboratory reagent. It is a primary antibody that can be used for research purposes. The core function of this product is to bind to a specific target molecule, enabling detection or quantification of that target in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Ab14705, by Abcam (2 mentions)
Ab14745, by Abcam (2 mentions)
Ab110252, by Abcam (2 mentions)
Ab14748, by Abcam (2 mentions)
Ab14734, by Abcam (2 mentions)
Ac 74, by Merck Group (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Uqcrc2, by Abcam (1 mentions)
Ab119686, by Abcam (1 mentions)
Ab23707, by Abcam (1 mentions)
Ab169540, by Abcam (1 mentions)
Ab106814, by Abcam (1 mentions)
Ab155999, by Abcam (1 mentions)
Ab110413, by Abcam (1 mentions)
Ab51520, by Abcam (1 mentions)
Ab189945, by Abcam (1 mentions)
Gtx101098, by GeneTex (1 mentions)
β actin a5441, by Merck Group (1 mentions)
Ab14715, by Abcam (1 mentions)
Ab76021, by Abcam (1 mentions)
7 protocols using ab14746
1
siRNA Knockdown of Mitochondrial Enzymes
2
Isolation and Analysis of Mitochondrial Complexes
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Mitochondria were isolated from cells by differential centrifugation and then resuspended in 10 μl of a buffer containing 50 mm Bis-Tris and 1 m 6-aminocaproic acid2 (link),54 (link). Digitonin (digitonin/protein ratio of 4 g g−1) was added to solubilize the mitochondria. After a 30 min incubation at 4 °C, the samples were centrifuged at 22,000 × g and the solubilized proteins were obtained from the supernatant. The solubilized proteins were then supplemented with 1 μl of sample buffer (5% Coomassie Brilliant Blue G-250 in 0.5 m 6-aminocaproic acid). BN-PAGE was performed as described previously2 (link) and subsequent immunoblotting was performed according to standard protocols. The blots were probed with anti-NDUFA9 (1:5000 dilution, 459100, Invitrogen), anti-RISP (1:5000 dilution, ab14746, Abcam), anti-COX1 (1:5,000 dilution, ab14705, Abcam), Fp70 (1:5000 dilution, 459200, Invitrogen), and anti-COX7RP antibodies (1:3000 dilution)2. Signal intensities for mitochondrial respiratory complexes and supercomplexes were quantified by normalization with their corresponding Fp70 using triplicated western blot analysis.
3
Mitochondrial Respiratory Complex Quantification
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Immunoblot analysis was used to assess protein levels of subunits of complexes I, III and IV, and subunits of F0F1-ATPase in renal cortical mitochondria using 20 μg of mitochondrial protein, as described previously [57 (link),61 (link)]. Antibodies against NDUFA9, NDUFS3, and NDUFB7 subunits of complex I (Cat #A21344, A21343, and 21359) were purchased from Molecular Probes/ThermoFisher Scientific (Waltham, MA, USA). Antibodies against α- and γ-subunits of F0F1-ATPase (Cat #ab14748 and #ab119686, respectively) and the core protein 1 (UQCRC1), core protein 2 (UQCRC2), and Rieske protein subunits of complex III (Cat #ab110252, #ab14745, and #ab14746, respectively) were supplied by Abcam (Cambridge, MA, USA). Antibodies against β-subunit of F0F1-ATPase (Cat #A21351) were supplied by Life Technologies Corporation (Carlsbad, CA, USA). Cytochrome oxidase subunit I antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat #sc-58347). Citrate synthase levels served as the loading control using the citrate synthase antibody (Cat #D7V8B) supplied by Cell Signaling Technology (Danvers, MA, USA).
4
Comprehensive Mitochondrial Protein Analysis
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The following specific antibodies were used for Western blot analysis: anti-UQCRFS1 (1:1000 dilution, ab14746, Abcam), anti-NDUFS1 (1:1000, ab169540, Abcam), anti-VDAC1 (1:3000, ab14734, Abcam), OXPHOS cocktail (1:1000, ab110413, Abcam), anti-LC3 (1:1000, ab51520, Abcam), anti-PINK1 (1:500, ab23707, Abcam), anti-parkin (1:500, #4211, Cell Signaling Technology), anti-ubiquitin (1:1000, P4D1, BioLegend), anti-PGC-1α (1:1000, ab106814, Abcam), anti-PPARα (1:1000, GTX101098, GeneTex), anti-TFAM (1:1000, GTX103231, GeneTex), anti-ATP5A (1:1000, ab14748, Abcam), anti-SDHA (1:1000, GTX101689, GeneTex), anti-SDHB (1:2000, GTX104628, GeneTex), anti-SDHC (1:500, ab155999, Abcam), and anti-SDHD (1:500, ab189945, Abcam) antibodies.
5
Western Blotting of Liver Proteins
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Western blotting analyses on whole liver protein samples were performed as previously described [31 (link)]. The separated proteins on the PVDF membranes were detected using the following antibodies: mouse monoclonal antibodies against ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 (anti-Uqcrfs1 [5A5] ab14746), ubiquinol-cytochrome C reductase core protein I (anti-Uqcrc1 [16D10AD9AH5] ab110252), ubiquinol-cytochrome C reductase core protein II (anti-Uqcrc2 [13G12AF12BB11] ab14745) from Abcam (Cambridge, MA, USA) and mouse monoclonal antibody β-actin (A5441) from Sigma-Aldrich (Oakville, ON, Canada).
6
Antibody Characterization for Cell Biology Research
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Antibodies used in this study include antiactin (MAB1501; EMD Millipore), anti–α-adaptin (sc-17771; Santa Cruz Biotechnology, Inc.), anti-CD63 (556019; BD), anti-DNA (61014; Progen), anti-Drp1 (611113; BD), anti-Eps15 (sc-534; Santa Cruz Biotechnology, Inc.), anti-Flag (anti-DDDDK, ab1257; Abcam), anti-Flag (F1804; Sigma-Aldrich), anti-GFP (ab6673; Abcam), anti-GFP (A6455; Invitrogen), anti-LAMP2 (sc-18822; Santa Cruz Biotechnology, Inc.), anti-LBPA (Z-SLBPA; Eschelon), anti-parkin (sc-32282; Santa Cruz Biotechnology, Inc.), anti-PDH E2/E3bp (ab110333; Abcam), anti-PINK1 (6946; Cell Signaling Technology), anti-PMP70 (SAB4200181; Sigma-Aldrich), anti-SDHA (ab14715; Abcam), anti-SNAP29 (ab138500; Abcam), anti-Stx17 (17815–1-AP; ProteinTech), anti-Stx17 (HPA001204; Sigma-Aldrich), anti-TIM23 (611222; BD), anti-TIP47 (Novus Biologicals), anti-TOM20 (sc-11414; Santa Cruz Biotechnology, Inc.), anti-UQCRFS1 (referred to herein as CIII-Rieske, ab14746; Abcam), anti-VAMP7 (sc-166394; Santa Cruz Biotechnology, Inc.), anti-VAMP8 (ab76021; Abcam), anti-VDAC1 (ab14734; Abcam), and anti-Vps41 (ab181078; Abcam). Unless otherwise specified, all reagents were purchased from Sigma-Aldrich. The TIP47 antibody was a gift from P. McPherson (McGill University, Montreal, Quebec, Canada).
7
Quantitative Protein Expression Analysis
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