The Parkin‐siRNA lentivirus and control lentivirus were constructed by Genechem. 3,4‐dihydro‐5‐[4‐(1‐piperidinyl)butoxy]‐1(2H)‐isoquinoline (DPQ) was from Apexbio (Houston, USA). Cyclosporin A and carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP) was from Selleck. N‐acetyl cysteine was from Beyotime (S0077). Anti‐PAR, Anti‐PARkin, anti‐COX IV, anti‐cyclophilin D (CypD) and anti‐translocator protein (TSPO) was from Abcam. Anti‐poly(ADP‐ribose) polymerase 1 (PARP‐1) was from Proteintech. Secondary antibodies for Western blotting were from Cell Signaling Technology.
Anti cox 4
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-COX IV is a laboratory reagent used to detect the presence and quantify the levels of the cytochrome c oxidase subunit IV (COX IV) protein in biological samples. COX IV is a key component of the mitochondrial electron transport chain and is commonly used as a marker for mitochondrial content in cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Abcam (4 mentions)
Anti gapdh, by Cell Signaling Technology (4 mentions)
Anti β actin, by Cell Signaling Technology (4 mentions)
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti lc3b, by Cell Signaling Technology (3 mentions)
Anti β actin, by Abcam (3 mentions)
Anti sdha, by Abcam (3 mentions)
Anti coxi, by Abcam (3 mentions)
Anti cytc, by Abcam (3 mentions)
Anti mfn2, by Abcam (3 mentions)
Anti parkin, by Abcam (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Anti cleaved caspase 9, by Abcam (2 mentions)
Anti gsk3β, by Abcam (2 mentions)
Anti cleaved caspase 3, by Abcam (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Anti bcl 2, by Abcam (2 mentions)
Cell mitochondria isolation kit, by Beyotime (2 mentions)
Fluorescent secondary antibody, by Cell Signaling Technology (2 mentions)
44 protocols using anti cox 4
1
Parkin-siRNA Lentivirus Impacts Mitochondrial Function
2
Mitochondrial Enzyme Profiling in Ercc1 Mice
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Liver and kidney samples from 18-week-old Ercc1−/Δ and WT mice (n = 5) were homogenized in RIPA buffer (Pierce, Rockford, IL) with protease inhibitor cocktail (Roche, Indianapolis, IN). Mitochondrial extracts were prepared using Mitochondria Isolation Kit (Pierce) per the manufacturer’s specifications. Samples were separated on 4–20% polyacrylamide gel (Bio-Rad, Hercules, CA), transferred to nitrocellulose membrane, blocked and blotted with anti-PCNA (PC10, Santa Cruz Biotechnology, Santa Cruz, CA), anti-ERCC1 (D-10, Santa Cruz Biotechnology), anti-COXIV (Abcam, Cambridge, MA), anti-XPF (SPM228, Novus Biologicals, Littleton, CO) or anti-GAPDH, anti-MnSOD, anti-CuZnSOD, anti-catalase (3H3L29), anti-XO, and anti-rabbit secondary (all from Life Technologies, Carlsbad, CA) then visualized with ECL reagent (Pierce). Films exposed to membrane were imaged with ImageJ (NIH, Bethesda, MD). GAPDH was used as a loading control.
3
Immunoblotting for Mitochondrial Proteins
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Mouse monoclonal anti-mitofusin 2 (Cat # ab56889 - 1:1000, RRID:AB_2142629 ), anti-COX-IV (Cat #ab16056 - 1:1000, RRID:AB_443304 ) and anti-GAPDH (Cat #ab8245 - 1:1000, RRID:AB_2107448 ) were from AbCAM (Cambridge, MA, USA). Rabbit polyclonal anti-Stathmin-2/Superior Cervical Ganglion 10 (SCGN10; Cat # NBP1-4946, RRID:AB_10011569 ) was from Novus Biologicals (Littleton, CO, USA).Rabbit polyclonal FSP-1 was from Sigma Millipore (Cat # 07–2274, RRID:AB_10807552 ). Anti-mouse monoclonal -MNX1was from DSHB (1:10, Cat# 81.5C10, RRID:AB_2145209 ). Mouse monoclonal anti-β -tubulin III (Cat # 801201- 1:500, RRID:AB_2313773 ) was from Biolegend (San Diego, CA, USA). Peroxidase-conjugated anti-mouse IgG (Cat #7076S - 1:1000, RRID:AB_330924 ) was from Cell Signaling (Danvers, MA, USA). Goat anti-rabbit IgG (Spicier reactivity Goat, Host/Isotype Rabbit/IgG; Cat #31460, RRID:AB_228341 ) and Alexa-Fluor 488 anti-mouse ThermoFisher (Waltham, MA, USA Cat #A11008, RRID:AB_143165 ). α-Bugarotoxin Alexa flour 594 was from ThermoFisher, Waltham, MA, USA Cat:# B12423.
4
SINE Compounds Inhibit Nuclear Export
5
Antibody Profiling for Focal Adhesion
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Antibodies used were as follows: anti-Paxillin (RRID:AB_647289 ), anti-GM130 (RRID:AB_398141 ), anti-p130Cas (RRID:AB_397667 ) and anti-RACK1 (RRID:AB_397577 ) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID:AB_2122095 ) (Abcam, Cambridge, UK), anti-CoxIV (RRID:AB_10694213 ), anti-FAK (RRID:AB_10694068 ), anti-pFAK Y397 (RRID:AB_2173659 ), anti-pPaxillin Y118 (RRID:AB_2174466 ), anti-Rab7 (RRID:AB_1904103 ), anti-pSrc Y416 (RRID:AB_331697 ), anti-Src (clone 36D10) (RRID:AB_10693939 ), anti-LC3B (RRID:AB_2137707 ) and anti-GAPDH (RRID:AB_10622025 ) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID:AB_10842517 ), anti-Ambra1 (RRID:AB_2636939 ) and anti-pSrc Y416 (RRID:AB_309898 ) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID:AB_310789 ) (Millipore, Billerica, MA, USA). LC3B antibody was from MBL (RRID:AB_1279144 ) (MBL International, Woburn, MA, USA). TRITC-phalloidin was purchased from Life Technologies (RRID:AB_2572408 ) (Paisley, UK). Anti-rabbit (RRID:AB_2099233 ) or mouse (RRID:AB_330924 ) peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technologies. Dasatinib was obtained from Bristol Myers Squibb (Princeton, NJ, USA) and PF562271 from Pfizer (Groton, CT, USA).
6
Immunofluorescence Labeling of MTERF3 and COX IV
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Immunofluorescence experiments were performed with standard protocols using the following antibodies to label MTERF3 and COX IV: anti-MTERF3 (1:200; Abcam), and anti-COX IV (1:200; Abcam). Secondary antibodies used were Alexa Fluor 594-labeled donkey anti-rabbit IgG (1:300; Life Technologies, Carlsbad, USA) and Alexa Fluor 488-labeled rabbit anti-mouse IgG (1:2500; Life Technologies).
7
Subcellular Fractionation of C2C12 Cells
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C2C12 cells were homogenized in ice-cold buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 250mM sucrose, 1 mM DTT, 0.5 mM PMSF, 20 mg/ml leupeptin, 20 mg/ml aprotinin, and 20 mg/ml trypsin inhibitor, using a Dounce homogenizer. After centrifugation at 800 g for 15 min at 4 °C the supernatant, free of debris and nonhomogenized cells, was further centrifuged at 10 000 g for 30 min at 4 °C to yield the mitochondrial pellet. The remaining supernatant was called cytosolic supernatant. Pellets were resuspended in lysis buffer and protein concentration of the fractions was estimated. Protein fraction extracts were resolved by 15% SDS-polyacrylamide gel electrophoresis and western blot assays were performed as described before. Membranes were incubated with specific antibody against Bax, (Cell Signaling Technology Inc, Beverly, MA). Cross contamination between fractions was assessed by western blot analysis using anti-COX IV (Abcam) as mitochondrial marker.
8
Western Blot Analysis of Apoptosis Markers
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Proteins were extracted from cell lysates using RIPA lysis Buffer and the protein concentration was measured using the Enhanced BCA Protein Assay Kit (Beyotime) following to the manufacturer’s recommendations. Proteins were denatured by heat, and then separated by SDS-PAGE electrophoresis, finally transferred to PVDF membranes. The membranes were blocked using 1% bovine serum albumin solution for 1 h at room temperature, and then incubated with primary antibodies, including anti-cytochrome c (1:1000; Abcam), anti-cleaved caspase-9 (1:1000; Abcam), anti-cleaved caspase-3 (1:1000; Abcam), anti-phospho-Akt (1:1000; Abcam), anti-Akt (1:1000; Abcam), anti-phospho-Gsk-3β (1:1000; Abcam), anti-Gsk-3β (1:1000; Abcam), anti-phospho-Stat-3 (1:1000; Abcam), anti-Stat-3 (1:1000; Abcam) anti-COXIV (1:1000; Abcam) and anti-β-actin (1:1000; Zhongshan Jinqiao Biotechnology, Beijing, China) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibody (1:5000; Zhongshan Jinqiao Biotechnology, Beijing, China) at room temperature for 30 min. Protein bands were detected using a BeyoECL Plus Kit (Beyotime) following to the manufacturer’s protocols. Relative densitometry was analyzed using Image J2x analysis software (NIH, U.S.A.).
9
Western Blot Analysis of Protein Expression
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Western blot analysis was performed as previously described (10 (link)). Briefly, equal amounts of protein (20 µg) were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Millipore, Sigma). The membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) in Tris-buffered saline Tween-20 (0.1%) for 2 h at room temperature and then incubated with primary antibodies at 4˚C overnight. The following primary antibodies were used: Anti-PHB (dilution, 1:500; product code ab75766; Abcam), anti-COX IV (dilution, 1:700; product code ab202554; Abcam), anti-cleaved-caspase 3 (dilution, 1:400; product no. 9661; Cell Signaling Technology, Inc.), anti-cleaved PARP (dilution, 1:500; product no. 5625; Cell Signaling Technology, Inc.) and anti-β-actin (dilution, 1:1,000; cat. no. sc-8432; Santa Cruz Biotechnology, Inc.). Immunoreactive proteins were detected with horseradish peroxidase-conjugated secondary antibodies (dilution, 1:5,000; cat. no. sc-2357; Santa Cruz Biotechnology, Inc.). Quantification was performed according to the NIH Image version-1.61 software (National Institutes of Health).
10
Western Blot Analysis of Protein Expression
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