The primary antibodies used were the anti‐Ubiquitin (Santa Cruz Biotechnology, Inc., sc‐8017), anti‐foxo (COSMO BIO CO, CAC‐THU‐A‐DFOXO), anti‐26S proteasome α (Santa Cruz Biotechnology, Inc., sc‐65,755), anti‐26S Proteasome p54 (Rpn10) (Santa Cruz Biotechnology, Inc., sc‐65,746), anti‐pAMPK (Cell Signaling Technology, #2535), anti‐GABARAP (Atg8a) (Cell Signaling Technology, #13733), anti‐Lamp1 (Abcam, ab30687), anti‐ATP5A (Abcam, ab14748), anti‐Actin (Cell Signaling Technology, #8457), and anti‐Gapdh (Sigma‐Aldrich, G9545). The anti‐ref(2)P/p62 antibody was kindly donated from Prof. G. Juhász. The secondary antibodies Peroxidase AffiniPure Donkey anti‐Mouse IgG (715‐035‐150) and Peroxidase AffiniPure Donkey anti‐Rabbit IgG (711‐035‐152) were purchased from Jackson ImmunoResearch Laboratories, Inc. The anti‐Rabbit‐IgG Alexa Fluor 647 (711‐605‐152), anti‐Rabbit‐IgG Alexa Fluor 488 (111‐545‐003), anti‐Mouse‐IgG Rhodamine (TRITC) AffiniPure (715‐025‐151), anti‐Mouse‐IgG Alexa Fluor 488 (115‐545‐003), and anti‐Mouse IgG DyLight™ 405 (715‐475‐151) antibodies were also from Jackson ImmunoResearch Laboratories, Inc. Ponceau S solution (6226–79) was from Sigma‐Aldrich.
Anti atp5a
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-ATP5A is a primary antibody that targets the ATP synthase subunit alpha (ATP5A). ATP5A is a key component of the mitochondrial ATP synthase complex, which is responsible for the production of ATP, the primary energy currency of the cell. This antibody can be used to detect and quantify the expression of ATP5A in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti tom20, by Santa Cruz Biotechnology (4 mentions)
Anti gapdh, by Merck Group (3 mentions)
Anti gfap, by Merck Group (3 mentions)
Anti uqcrc2, by Abcam (3 mentions)
Anti gapdh, by Abcam (3 mentions)
Anti hsp60, by Santa Cruz Biotechnology (3 mentions)
Anti vinculin, by Merck Group (2 mentions)
Anti darpp32, by Abcam (2 mentions)
Anti htt epr5526, by Abcam (2 mentions)
Hdac1, by Abcam (2 mentions)
Anti polyq php3, by Abcam (2 mentions)
Anti pde10a, by Abcam (2 mentions)
Anti polyq mw1, by Merck Group (2 mentions)
Anti htt mw8, by Developmental Studies Hybridoma Bank (2 mentions)
Anti sdhb, by Abcam (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Restore plus western blot stripping buffer, by Thermo Fisher Scientific (2 mentions)
Anti poly adp ribose polymerase parp, by Cell Signaling Technology (2 mentions)
Pseudokinase mlkl phospho s345, by Abcam (2 mentions)
Anti hsp70, by BD (2 mentions)
32 protocols using anti atp5a
1
Immunoblotting for Autophagy Markers
2
Antibodies and Dilutions for Huntington's Disease
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The following antibodies and dilutions were used in this study: Anti-HTT Ab1 (aa1–17,1 (link)) 1:50 for capillary immunoassay and 1:2000 for western blot; anti-HTT EPR5526 (Abcam, Waltham, MA, ab109115, 1:2000 for western blot); anti-polyQ MW1 (MilliporeSigma, Burlington, MA, MABN2427, 1:50 for capillary immunoassay); anti-polyQ PHP3 (generous gift from Dr. Ali Khoshnan, 1:2000 for western blot); Anti-PDE10A (Abcam, Waltham, MA, #ab177933, 1:2000 for western blot); Anti-DARPP32 (Abcam, #ab40801, 1:2000 for capillary immunoassay); Anti-GFAP (MilliporeSigma, Burlington, MA, AB5804, 1:3000 for capillary immunoassay); Anti-GAPDH (MilliporeSigma, Burlington, MA, #MAB374, 1:10000 for western blot); Anti-Sodium channel subunit beta-4 (Abcam, Waltham, MA, #ab80539, 1:500 for western blot); Anti-vinculin (Sigma, St. Louis, MO, #V9131, 1:5000 for capillary immunoassay, 1:2000 for western blot); Anti-ATP5A (Abcam, Waltham, MA, #ab14748, 1:2000 for western blot); Anti-HTT MW8 (University of Iowa Developmental Studies Hybridoma Bank, 1:1000 for filter trap); Anti-HTT S830 (generous gift from Dr. Gillian Bates, 61 (link) 1:8000), HDAC1 (Abcam, Waltham, MA, ab32369–7, 1:4000).
3
Quantitative Analysis of Oxidative Stress
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Ten male flies were used for each condition. The antibodies used in Western blot analysis were as follows: anti-4HNE (1:1000, JalCA); anti-ATP5A (1:100,000, Abcam); and anti-tubulin (1:10,000, Sigma). HRP-conjugated anti-rabbit or anti-mouse IgG antibodies (Cell Signaling Technology) were used as secondary antibodies. Blots were visualized using Chemi-Lumi One Super reagent (Nacali Tesque Inc.). To quantify 4-HNE and ATP5A levels, staining intensity of each lane was measured using ImageJ (http://imagej.nih.gov/ij/ ). Measured area is indicated in the box.
4
Western Blot Analysis of ATP5A and Actin
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The pellet or supernatant from the previous centrifugation were mix NuPAGE LDS sample buffer (Thermo Fisher #NP0007) and heated at 70°C for 10 min. Samples were loaded onto NuPAGE 4% to 12% Bis-Tris Protein Gels (Thermo Fisher #NP0323BOX) and run with NuPAGE MES SDS running buffer (Thermo Fisher #NP0002). After semi-dry transfer, PVDF membrane (Millipore, Burlington, Massachusetts, USA, #IPVH00010) was blocked with 5% nonfat milk, and probed with anti-ATP5A (Abcam, Cambridge, United Kingdom, ab14748) or anti-actin (Abcam #ab3280) primary antibodies. The membrane was developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher #34096) and visualized by Amersham Hyperfilm (GE Healthcare, Chicago, Illinois, USA, #28906845).
5
Subcellular Fractionation and Western Blot Analysis
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The isolated ER, c-mito, p-mito and MAM fractions were resuspended in lysis buffer, separated on SDS-polyacrylamide gel, and blotted onto PVDF membranes. Western blots were probed with antibodies against specific protein markers to verify the purity of the fractions and the presence of frataxin. The primary antibodies used were anti-ATP5A (Abcam, Cambridge, United Kingdom, ab110273), anti-Sigma1R (Sigma-Aldrich, St. Louis, Missouri, U.S., HPA018002), anti-GRP75 (Santa Cruz Biotechnology, Dallas, Texas, U.S., sc-13967), anti-VDAC1 (Abcam, Cambridge, United Kingdom, ab14734) and anti-FXN (Abcam, Cambridge, United Kingdom, ab110328). Secondary antibody (anti-mouse or anti-rabbit IgG) coupled to peroxidase was used for detection of the reaction with Amersham ECL Prime (GE Healthcare, Chicago, Illinois, U.S.). Imaging was performed with an ImageQuant LAS4000 (GE Healthcare, Chicago, Illinois, U.S.).
6
Mitochondrial Antioxidant and OXPHOS Protein Analysis
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Total protein concentration was determined using the Bradford assay (Cell Signaling Technology, Denver, MA, USA). Samples were resolved in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to 0.45-μM polyvinyl difluoride membranes, and analyzed separately. After blocking with 5% skim milk at room temperature for 60 minutes, the blots were surveyed with primary antibodies. Expression of mitochondrial antioxidant was detected using anti-Mn-SOD (Abcam, Cambridge, MA, USA). We used anti-NDUFA9 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-SDHB (Abcam), anti-UQCRC2 (Abcam), anti-COXIV (Santa Cruz Biotechnology), and anti-ATP5A (Abcam) to detect the expression of oxidative phosphorylation complexes.
7
Western Blot Analysis of Protein Markers
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Total protein from each sample (10–20 μg) was separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Abs used for protein detection were the following: anti–poly(ADP-ribose) polymerase (PARP) (product no. 9542S; Cell Signaling Technology, Danvers, MA), anti–mixed lineage kinase domain-like pseudokinase (MLKL) phospho-S345 (product no. ab196436; Abcam), anti-GAPDH (product no. ab181602; Abcam), anti-HSP70 (product no. 610607; BD Biosciences), and anti-ATP5a (product no. ab176569; Abcam). Western blot membranes were stripped of Abs using Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific) when additional Ab probing was required.
8
Mitochondrial Function and Oxidative Stress
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5‐ALA hydrochloride (neo ALA Co. Ltd, Tokyo, Japan) and SFC (Komatsuya Corporation, Osaka, Japan) were provided by SBI Pharmaceuticals Co., Ltd. (Tokyo, Japan) Phalloidin (Sigma‐Aldrich, St. Louis, MO, USA), TOPRO‐3 (Invitrogen, Waltham, MA, USA), anti‐ATP5A (Abcam, Cambridge, UK), MitoTracker Deep Red FM (Thermo Fisher, Waltham, MA, USA), MT‐1 MitoMP Detection Kit (DOJINDO LABORATORIES, Kumamoto, Japan), anti‐4‐hydroxynonenal (4‐HNE; Abcam), Alexa Fluor 488 anti‐mouse IgG (Thermo Fisher), Alexa Fluor 488 anti‐rabbit IgG (Thermo Fisher), sucrose, Tris, MgCl2, aminohexanoic acid, Bis‐Tris, Coomassie Blue G (FUJIFILM Wako Pure Chemical, Osaka, Japan), Triton X‐100 (Sigma), anti‐citrate synthase (CISY11‐A; Alpha Diagnostics, San Antonio, TX, USA), anti‐rabbit IgG HRP‐Linked Whole Ab Donkey (GE Healthcare, Chicago, IL, USA), and ImmunoStar LD (FUJIFILM Wako Pure Chemical) were purchased.
9
Western Blot Analysis of Cellular Signaling
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Tissues were homogenized using TissueLyzer II (Qiagen, Hilden, Germany). Cells and tissues were lysed using the radioimmunoprecipitation assay (RIPA) buffer (30 mM Tris [pH 7.5], 150 mM sodium chloride, 1 mM sodium phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1% Nonidet P-40, 10% glycerol, and phosphatase and protease inhibitors). Western blot analyses were performed with 40 μg of protein, using commercially available antibodies. Anti-CHOP, anti-pERK, anti-ERK, anti-IRE1α, anti-pIRE1α, anti-pSMAD2/3, and anti-β-actin antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-NDUFA, anti-SDHA, anti-UQCR2, and anti-ATP5A antibodies were purchased from Abcam (Cambridge, UK). Anti-COL1A2, anti-COL1A1, anti-ATF4, anti-TGF-β1, and anti-IL-1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL; Bio-Rad, Hercules, CA, USA). Images were scanned using the Odyssey imaging system and quantified using Image Studio Digits (LI-COR Biosciences, Lincoln, NE, USA).
10
Mitochondria Isolation and Western Blot Analysis
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INS-1E cells [17 (link)] were cultured at 37 °C in a humidified atmosphere (5% CO2) in RPMI-1640 medium (Invitrogen) containing 11 mM glucose, supplemented with 10 mM Hepes (pH 7.3), 10% (v/v) heat-inactivated fetal calf serum (FCS; Brunschwig AG, Basel, Switzerland), 1 mM sodium pyruvate, 50 μM β-mercaptoethanol, 50 μg/mL penicillin, and 100 μg/mL streptomycin (INS-1 medium). Culturing HeLa cells has been previously described [18 (link)].
Mitochondria were isolated from INS1E and HeLa cell culture as previously described [19 (link)]. Western blots were previously described [20 (link)]. The membranes were probed with anti-MCU (Sigma-Aldrich, Buchs, Switzerland), anti-Tom20 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-ATP-5A (Abcam, Cambridge, United Kingdom) antibodies.
Mitochondria were isolated from INS1E and HeLa cell culture as previously described [19 (link)]. Western blots were previously described [20 (link)]. The membranes were probed with anti-MCU (Sigma-Aldrich, Buchs, Switzerland), anti-Tom20 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-ATP-5A (Abcam, Cambridge, United Kingdom) antibodies.
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