Cells were prepared as described in ref. 44 (link). Briefly, whole-cell pellets were solubilized in phosphate buffered saline, 1% dodecyl-maltoside (DDM), 1 mM PMSF (phenylmethylsulfonyl fluoride), and complete protease inhibitor (Thermo Fisher). Protein concentrations were measured by the Bradford assay (BioRad). Equal amounts of proteins were separated by Tris-Glycine SDS-PAGE. Membranes were blocked in TBST with 1% milk at room temperature for 1 h then primary antibodies (Proteintech Group: MTO1 (15650-1AP, 1:5000); TRMT61B (26009-1AP, 1:2000); MT-CYB (55090-1-AP, 1:1000); MT-ATP6 (55313-1-AP, 1:2000); MRPL11 (15543-1-AP, 1:20000); MRPL45 (15682-1-AP, 1:5000); MRPS27 (17280-1-AP, 1:5000). Abcam: MT-CO1 (1D6E1A8, 1:1000); SDHA (2E3GC12FB2AE2, 1:10000). Santa Cruz: TOM40 (sc-11414, 1:2000)) were incubated overnight at + 4 °C in 5% BSA/TBST and detected the following day with secondary HRP conjugates (Jackson ImmunoResearch) using ECL with film. Representative data of independent experiments were cropped in Photoshop with only linear corrections applied.
Mtco1
Manufactured by Abcam
Sourced in United Kingdom
MTCO1 is a protein that is a subunit of the cytochrome c oxidase complex, which is the last enzyme in the respiratory electron transport chain of mitochondria. It is essential for the proper function of the mitochondrial electron transport chain and cellular respiration.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Hsp60, by Enzo Life Sciences (3 mentions)
Oxphos cocktail, by Abcam (3 mentions)
Uqcrc2, by Abcam (3 mentions)
Any kd mini protean tgx stain free precast gels, by Bio-Rad (2 mentions)
Mt atp8, by Santa Cruz Biotechnology (2 mentions)
Stain free imaging system, by Bio-Rad (2 mentions)
Catch and release v2.0 kit, by Merck Group (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Sirt3, by Cell Signaling Technology (2 mentions)
Hsp60, by BD (2 mentions)
Sirt3, by Merck Group (2 mentions)
Anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Foxo3a, by Cell Signaling Technology (2 mentions)
Vdac1, by Abcam (2 mentions)
C300 imaging system, by Azure Biosystems (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Anti gapdh 14c10, by Cell Signaling Technology (2 mentions)
27 protocols using mtco1
1
Western Blot Analysis of Mitochondrial Proteins
2
Mitochondrial Protein Expression Analysis
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Whole cell lysates were collected using NP-40 or RIPA buffer with protease inhibitor cocktail (ThermoFisher Scientific, NY, #78443). 30mg total protein was separated by SDS–PAGE using Any kD™ Mini-protean TGX stain-free™ precast gels (BioRad, CA, #4568123), and transferred to PVDF membranes using the Trans-Blot Turbo™ Transfer System (BioRad, CA, #1704155). Membranes were incubated overnight with antibodies against DGUOK (Santa Cruz, #sc-398093, 1:200), HSP90β (Abcam, #ab32568, 1:100000), MT-ATP8 (Santa Cruz, #sc-84231, 1:200), cytochrome b (Santa Cruz, #sc-11436, 1:200), ND6 (Santa Cruz, #sc-20510, 1:200), MTCO1 (abcam, #ab14705, 1:1000), oxidative phosphorylation antibody cocktail (UQCRC2, ATP5A, MTCO1) (Abcam, #ab110413, 1:250), PGC1 alpha (Abcam, #ab77210, 1:400), SOD2 (Abcam, #13533, 1:5000), PINK1 (Abcam, #23707, 1:500) or Acetyl lysine (Abcam, #ab190479, 1:500) at 4°C. HRP-conjugated secondary antibodies were used at a dilution of 1:2000. Protein levels were calculated using BioRad stain-free Imaging System and were normalized to total protein using Image Lab software from BioRad. Immunoprecipitation was performed using Catch and Release® V2.0 kit (EMD Millipore, CA, #17-500) following the manufacturer’s directions.
3
Western Blot Analysis of Mitochondrial Proteins
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Cells, washed with PBS, or pulverized flash frozen tissue was lysed in cold NP-40 lysis buffer with protease inhibitors (50 mM Tris, pH 7.5, 250 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 50 mM NaF, 0.2 mM Na3 VO4, 1 g/ml leupeptin, 1 g/ml pepstatin, 100 g/ml phenylmethylsulfonyl fluoride), sonicated for 5 s at 20% amplitude, and centrifuged at 14,000 rpm for 20 minutes at 4°C. Protein concentrations of lysates were assayed using the Bradford method (Bio-Rad Protein Assay). Equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membrane (GE Healthcare). Following blocking, membranes were probed with the following primary antibodies overnight at 4°C: SIRT3 (Cell Signaling 2627S), SIRT3 (EMD Millipore 07-1596), HSP60 (BD Transduction 611563), ATF5 (abcam ab184923), NRF1 (abcam ab55744), ClpP (abcam ab 124822), SOD1 (Santa Cruz sc-11407), FOXO3a (Cell Signaling 2497S), Actin (EMB Millipore MAB1501R), SOD2 (EMB Millipore 06-984), LC3 (MBL International PM036), SDHA (abcam ab14715), MTCO1 (abcam ab45918), GFP (Santa Cruz sc-9996), NRF2 (cell Signaling 12721). Blots were then probed with horseradish peroxidase conjugated anti-mouse (Jackson ImmunoResearch or KwikQuant) or anti-rabbit secondary antibodies (Thermo Fisher Scientific or KwikQuant) and detected using enhanced chemiluminescence (GE Healthcare or KwikQuant).
4
Quantifying Mitochondrial Oxidative Stress
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Immunohistochemical stains using the antibodies TOM20 (rabbit polyclonal, Santa Cruz Biotech., Dallas, TX; dilution 1:100), MTCO1 (mouse monoclonal, Abcam, Cambridge, MA; dilution 1:2000), and Anti-8 Hydroxyguanosine (mouse monoclonal, Abcam, Cambridge, MA; dilution 1:400) were performed on formalin-fixed, paraffin-embedded tissues using the manufactures’ protocols.
The stained sections were scanned using the Aperio ScanScope XT systems (Aperio Technologies, Vista, CA) at 20X magnification. Immunohistochemical expression was quantified in all digital slides with either nuclear (Anti-8 Hydroxyguanosine) or cytoplasmic (TOM20 and MTCO1) algorithms using ImageScope analysis software (version 12; Aperio Technologies, Inc) and an H–score was calculated (Luna, 1968 ).
The stained sections were scanned using the Aperio ScanScope XT systems (Aperio Technologies, Vista, CA) at 20X magnification. Immunohistochemical expression was quantified in all digital slides with either nuclear (Anti-8 Hydroxyguanosine) or cytoplasmic (TOM20 and MTCO1) algorithms using ImageScope analysis software (version 12; Aperio Technologies, Inc) and an H–score was calculated (Luna, 1968 ).
5
Western Blot Analysis of Mitochondrial Proteins
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Commercially available antibodies were used in western blots (WBs), for the detection of OPA1 and TIM44 (BD Transduction Laboratories, Franklin Lakes, New Jersey, USA), OMA1 (Novus Biologicals, Littleton, Colorado, USA), tubulin (DSHB, University of Colorado, Colorado, USA), HSP60 (Enzo Life Science, Farmingdale, New York, USA), MTCO1 and VDAC1 (Abcam, Cambridge, UK). Anti-AFG3L2 N-terminal (OriGene, Rockville, Maryland, USA) and an anti-AFG3L2 C-terminal antibody (previously generated in the lab)7 (link) were used to detect AFG3L2. Secondary antibodies used included Horseradish Peroxidase (HRP)-conjugated antimouse and antirabbit IgG (Amersham Bioscience, Buckinghamshire, UK). Chloramphenicol succinate sodium salt (Merck, KGaA, Billerica, Massachusetts, USA) was used at a concentration of 200 µg/mL for 24 hours. Carbonyl cyanide-4 -(trifluoromethoxy)phenylhydrazone (FCCP) (Merck) was used at a concentration of 10 µM for 10 min.
6
Immunohistochemical Analysis of Tissue Samples
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Tissue samples were fixed with 10% formaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. Sections were deparaffinized in xylene and then rehydrated by alcohol series. To examine the histology, sections were stained with hematoxylin and eosin (H&E). For immunohistochemistry, sections were first treated with 3% H2O2 to block the endogenous peroxidase, then incubated with IHC protein block solution (DAKO, Carpinteria, CA). Sections were then reacted with primary antibody at 4°C for overnight, then sequentially reacted with horseradish peroxidase-conjugated secondary antibody (DAKO). After washing, sections were incubated with diaminobenzidine tetrachloride solution and counterstained with Mayer’s hematoxylin. For double immunofluorescence staining, sections were incubated with primary antibodies, then incubated with fluorescence-conjugated secondary antibodies (Abcam, Cambridge, UK). Immunofluorescence signal was detected under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The following primary antibodies were used: Crif1 (Santa Cruz Biotechnology, Santa Cruz, CA), MTCO1 (Abcam), K15 (Abcam), K5 (Santa Cruz Biotechnology, Santa Cruz, CA), AE15 (Thermo Fisher Scientific), AE13 (Abcam), Lgr5 (Thermo Fisher Scientific) and Ki67 (Vector Laboratories, Burlingame, CA).
7
Synthesis and Characterization of Novel Anticancer Compounds
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Maritoclax (marinopyrrole A) and dimethoxymaritoclax were synthesized with purity >99% as previously described [31 (link)]. Dinaciclib was obtained from Stratech Scientific Ltd (Suffolk, UK). ABT-263 was obtained from Selleck Chemicals Co. (Houston, TX, USA). MitoQ was a kind gift from Prof. M. Jackson, University of Liverpool, UK. Antibodies against BIM, cleaved PARP, BCL-XL, BCL-w, MFN1 and MFN2 from Cell Signalling Technology Inc (Danvers, MA, USA), BCL-2 from Dako (Ely, UK), BFL-1 (a kind gift from Prof. J. Borst, The Netherlands Cancer Institute, Amsterdam, The Netherlands), ROMO1 from OriGene (Rockville, MD, USA), cytochrome c, DRP1 and OPA1 from BD biosciences (Oxford, UK), SDHB, UQCRC2, MTCO1, and ATPB from Abcam (Cambridge, UK), NOXA and tubulin from Calbiochem (Nottingham, UK), MCL-1, GAPDH, NDUFA9 and NDUFB8 from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and BAK from Millipore (Watford, UK) were used. MitosoxRed was purchased from Molecular Probes, Inc. (Eugene, OR, USA). All other reagents, unless mentioned otherwise, were from Sigma-Aldrich Co. (St. Louis, MO, USA).
8
Immunofluorescence Staining of Cellular Markers
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Cells were fixed with 4% paraformaldehyde for 15 min and washed 3× with Tris-buffered saline (TBS) before the blocking solution consisting of TBS with 10% donkey serum, 0.1% Triton X-100 and 0.1% Tween-20 was applied for 1 h. All primary antibodies were diluted in blocking solution (MTCO2, Abcam; MTCO1, Abcam; NDUFS8, Abcam; NDUFS2, Abcam; GARS, Proteintech; S-100Beta, Abcam; Collagen 1, Abcam; Nestin, Milliopore; GRFS1, Abcam; eIF4E, Abcam; Mitotracker, Invitrogen). Incubation of the primary antibody was performed overnight at 4°C. The next day, cells were washed 3× in TBS before the secondary antibody (Alexa-Fluor Anti-Rabbit 488, Alexa-Fluor Anti-mouse 594, Invitrogen) and DAPI diluted in blocking solution was applied for 1.5 h at room temperature. Immunofluorescence images were collected using a Zeiss Axio Imager Z1 fluorescence microscope equipped with Zeiss Apotome 2 (Zeiss, Germany) in AxioVisionRel 4.9 software.
9
Mitochondrial Proteome Analysis in Frozen Tissue
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Frozen cortex tissue samples were lysed by mechanical homogenization with RIPA buffer containing protease and phosphatase inhibitors, and analyzed by SDS–PAGE and western blot. Subsequently, the concentration of extracted protein was determined by using the Bio-Rad Protein Assay. Proteins were detected using the following antibodies: HSP60 (Enzo Life Science), CLPP (Sigma), anti-GAPDH (14C10) (Cell Signaling), LONP1 (Sigma), PINK1 (Novus Biologicals), LC3 A/B (Cell Signaling), SDHB (Oxphos cocktail, Abcam), MTCO1 (Abcam), Ubiquitin (Enzo), P62 (BD Transduction Laboratories), Phopsho P62 (Cell Signaling), VDAC (Abcam). In addition to the housekeeping proteins, loading was monitored by Ponceau Red to ensure a homogeneous loading. Antibody detection reactions for all the immunoblot experiments were developed by enhanced chemiluminescence (Advansta) and imaged using the c300 imaging system (Azure Biosystems). Pixel intensity was quantified by using ImageJ software.
10
Antibody Panel for Protein Detection
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Primary antibodies employed in this study were α-tubulin (Sigma, T6074), HA (Roche Applied, Basel, Switzerland, 11583816001), ATF4 to probe human cell samples (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-22800), dATF4 to probe Drosophila samples (a kind gift from Min Kang, University of Ulsan, Seoul, South Korea), GAPDH (Sigma, G8795), NDUFS3/complex I (Abcam, Cambridge, UK, ab14711), mtCO1 (Abcam, ab90668), α-tubulin (Sigma, T6074) and TH (Immunostar, Hudson, WI, USA, 22941).
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