Western Blotting was carried out using specific antibodies; Bcl-2 (D17C4, Cell Signaling, France), Bax (ab115193, Abcam, France), aconitase 2 (ab110321, Abcam, France), nitrotyrosine (05-233, Millipore, France), 4HNE (ab5605, Millipore, France), sirtuine-3 (D22A3, Cell signaling, France), sirtuine-1 (SC-9475, Cell signaling, France), phosphorylated sirtuine-1 (Sc-2314, Cell signaling, France), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a, ab54481, Abcam, France), nuclear factor (erythroid-derived 2)-like 2 (NRF2, ab31163, Abcam, France), mammalian target of rapamycin (mTOR, 2983S, Cell signaling, France), phospho mTOR (5536P, Cell signaling, France), eNOS total (610296, BD Transduction, Le Pont de Claix, France) and eNOS phophorylated Ser1177 (C9C3; Cell-Signaling, Saint Quentin-Yvelines, France). Protein quantification level was expressed as ratio of total proteins loaded on precast gels with stain-free technology (BioRad, France). All blots were obtained by imaging with chemidoc and quantificated with ImageLab software (BioRad).
Ab110321
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab110321 is a lab equipment product offered by Abcam. It is designed to perform a specific function within a laboratory setting. The core function of this product is to enable users to carry out various experimental procedures and analytical tasks. However, a detailed description of its intended use or specific applications is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (1 mentions)
Nitrotyrosine, by Merck Group (1 mentions)
Ab5605, by Merck Group (1 mentions)
Ab31163, by Abcam (1 mentions)
Ab54481, by Abcam (1 mentions)
Bcl 2 d17c4, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ab14713, by Abcam (1 mentions)
Anti mouse hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidine difluoride pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Boster Bio (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Ab39012, by Abcam (1 mentions)
Ab126595, by Abcam (1 mentions)
Ab78066, by Abcam (1 mentions)
Ab84036, by Abcam (1 mentions)
Western ecl substrate kit, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ab32503, by Abcam (1 mentions)
Genelute gel extraction kit, by Merck Group (1 mentions)
8 protocols using ab110321
1
Western Blot Analysis of Protein Targets
2
Mitochondrial Protein Analysis in Rat Embryos
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Male and female heterozygous rats were mated and the WT and homozygous embryos were identified and same‐genotype embryos were combined for analysis. The combined embryo lysates of were separated on 12% SDS‐PAGE. Western blot was performed according to standard procedures. The primary antibodies for NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9, 1:1000, Abcam, ab14713), and aconitase 2 (ACO2, 1:1000, Abcam, ab110321) were visualized using anti‐mouse HRP‐conjugated secondary antibody (Santa Cruz Biotechnology, Inc.), and TOMM20 (1:250, Ivitrogen, PA5‐52843) was visualized using anti‐rabbit HRP‐conjugated secondary antibody (Santa Cruz Biotechnology, Inc.). Quantitative analysis was performed by densitometry using NIH Image software and normalized to TOMM20.
3
Quantification of Chondrocyte Proteins
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Briefly, chondrocytes were homogenized on ice and lysed with RIPA lysis buffer (Boster, Wuhan, China). After 30 minutes, the lysates were collected and centrifugated at 12 800 g for 20 minutes at 4℃. 25 μg proteins from each sample were separated by 10% SDS‐PAGE gel and then transferred to polyvinylidine difluoride PVDF membranes (pore size 0.45 μmol/L, Millipore, Billerica, MA), which were subsequently incubated with targeted primary antibodies anti MMP3 (17873‐1‐AP, proteintech, USA), MMP13 (ab39012, Abcam, USA), FIS1 (10956‐1‐AP, proteintech, USA), DRP1 (#8570, CST, USA), MFF (#84580, CST, USA), iron regulatory protein 1 (IRP1, ab126595, Abcam, USA), iron regulatory protein 2 (IRP2, ab110321, Abcam, USA), transferrin receptor (TfR1, ab84036, Abcam, USA), Ferroportin (FPN, ab78066, Abcam, USA), β‐actin (#BM0627, Boster, Wuhan) at 4°C overnight. After being washed for three times with TBST, membranes were incubated with respective secondary antibodies at room temperature for 1 hour. Bands and band density were detected using Western ECL Substrate Kit (Thermo Pierce, USA) and analyzed with the built‐in software of Bio‐Rad scanner (Bio‐Rad, Hercules, CA). Images were acquired with Bio‐Rad scanner (Hercules, CA) and densitometry was quantified by digital image analysis software (Quantity One, Bio‐Rad, Hercules, CA).
4
Cellular Signaling Pathways Modulation
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RPMI 1640 medium (61870-036) and fetal bovine serum [FBS] (10099141) were purchased from Gibco (USA, NY). CORT and neferine (HY-N0441) were purchased from MedChem Express (NJ, USA). The GenElute Gel Extraction Kit (NA1111) was purchased from Sigma-Aldrich (Darmstadt, Germany). BamHI (R0136S) and XhoI (R0146S) were purchased from NEB (NY, USA). Lipofectamine 3000 Transfection Reagent (L3000015) was purchased from Invitrogen (CA, USA). G418 (G8161) was purchased from Solarbio (Beijing, China). RNApure Tissue and Cell Kit (CW0584), HiFiScript cDNA Synthesis Kit (CW2569), and Super TaqMan Mixture (CW2698) were obtained from Cwbiotech (Beijing, China). Antibodies against Bcl-2 (ab32124), Bax (ab32503), Bad (ab32445), p53 (ab26), Bak (ab32371), succinate-CoA ligase GDP-forming beta subunit [SUCLG2] (ab96172), aconitase 2 [ACO2] (ab110321), malate dehydrogenase 1 [MDH1] (ab180152), citrate synthase [CS] (ab96600), isocitrate dehydrogenase [IDH] (ab172964), NF-κB p65 (ab16502), and glyceraldehyde 3-phosphate dehydrogenase [GAPDH] (ab8245) were purchased from Abcam (Massachusetts, US). ELISA kits for IL-1β (ab100562), IL-2 (ab174444), IL-6 (ab46027), TNF-α (ab181421), interferon-γ [IFN-γ] (ab46025), and granulocyte colony-stimulating factor [G-CSF] (ab100524) were purchased from Abcam. Rabbit and mouse secondary antibodies were purchased from ECL.
5
Prostate Cancer Tissue Microarray Analysis
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Tissue microarray (TMA) was constructed in collaboration with Shanghai Biochip Company (Shanghai, China) using tumorous (T) and adjacent non‐tumorous (ANT) tissues from 90 prostate cancer patients (180 cores). Immunohistochemistry (IHC) staining for ACO2 (ab110321; Abcam) expression was performed on 5 µm sections of the TMA. TMA slides were scanned using the Aperio slide scanner and analyzed by ImageScope software (Aperio). IHC staining was scored by two independent pathologists using the following scoring: proportion of positive stain (0, 0%; 1, 1‐25%; 2, 26‐50%; 3, 51‐75%; 4, >75%) and mean stain intensity (0 for absence of staining, 1 for weak staining, 2 for moderate staining, and 3 for strong staining). Final scores were calculated as the product of the proportion of positive stained cells and the mean stained intensity (range 0‐12). Samples with scores 0‐5 were considered ACO2 negative, and samples with scores 6‐12 were ACO2 positive.
6
Mitochondrial Protein Profiling by Immunoblot
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Cellular proteins (20 μg) solubilized in Laemmli buffer were resolved by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (GE Healthcare). For immune-detection, an anti-ACO2 monoclonal antibody (#ab110321, Abcam, 1/1000) was used. Monoclonal anti-VDAC and anti-α-tubulin antibodies were used as mitochondrial and cytosolic reference markers, respectively (Supplementary Fig. 1 ).
7
Mitochondrial Metabolism Analysis Protocol
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L-glutamine, dimethyl alpha-ketoglutaric acid, U-13C-glutamine, octyl-(R)-2HG, rotenone, oligomycin, and 2-[2-[4-(trifluoromethoxy)phenyl] hydrazinylidene]-propanedinitrile were purchased from Sigma-Aldrich (MO, USA). Fluorocitrate was prepared from DL-fluorocitric acid barium salt (Sigma-Aldrich) as previously described41 (link). Antimycin A was purchased from Abcam (MA, USA). Devimistat (CPI-613) was purchased from Selleck (TX, USA). MG132 was purchased from MCE (NY, USA). Antibodies recognizing β-actin (sc-47778; 1:5000), GAPDH (sc-32233; 1:1000), and NDUFS3 (sc-374282; 1:1000) were purchased from Santa Cruz Biotechnology (TX, USA). Antibodies recognizing IscU (#14812; 1:1000), NDUFSC1 (#12444-1-AP; 1:1000), and TOM40 (#18409-1-AP; 1:1000) were purchased from Proteintech (IL, USA). Antibodies recognizing UQCRFS1 (ab191078; 1:1000), aconitase 2 (ab110321; 1:1000), SDHB (ab14714; 1:1000), and α-KGDH (ab58724; 1:1000) were purchased from Abcam (Cambridge, United Kingdom). The secondary anti-mouse (#7076; 1:2000) or anti-rabbit (#7074; 1:2000) IgG antibodies and an anti-Myc antibody (#2276; 1:1000) were purchased from Cell Signaling Technology (MA, USA).
8
Western Blotting of Mitochondrial Proteins
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Isolated mitochondria from primary human skin fibroblasts were used for Western blotting. In brief, mitochondrial lysates were loaded onto a 10% SDS-PAGE gel. Electrophoresis was performed at 150 V for 35 min. Proteins were blotted onto nitrocellulose membranes (Transblot Turbo Pack; Biorad, Hercules, CA, USA) with the Transblot Turbo System from Biorad. Blocking was carried out using the Western blocking reagent (Roche, Basel, Switzerland) for 30 min at room temperature. Membranes were incubated with the following primary antibodies: mouse monoclonal anti-ACO2 antibody overnight at 4 °C (Abcam, Cambridge, UK; ab110321) and mouse monoclonal anti-VDAC1 antibody 1 h at room temperature (Abcam, ab14734). The secondary antibody from the DAKO Envision kit was used. The Lumi-Light Western blotting substrate (Roche) was used for development.
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