Cox 4

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, China, Netherlands

COX IV is a laboratory equipment product that functions as a cytochrome c oxidase subunit IV. It is part of the electron transport chain in mitochondria and plays a critical role in cellular respiration.

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Lab products found in correlation

201 protocols using cox 4

Comprehensive Western Blot and Immunostaining Protocol

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The following antibodies were used for western immunoblotting: PHB1 (70R-5543, Fitzgerald), PHB2 (14085, Cell Signaling), LC3 (L7543, Sigma), p62 (5114, Cell Signaling), Tim50 (sc-515268, Santa Cruz), CoxIV (4850, Cell Signaling), Nix (12396, Cell Signaling), FundC1 (ABC506, Millipore), Bnip3 (3769, Cell Signaling), Optineurin (100000, Cayman Chemical), NDP52 (H000 10241-B01P, Abnova), GST (sc-138, Santa Cruz), Parkin (MAB5512, Millipore), GFP (2956, Cell Signaling), Cytochrome C (4280, Cell Signaling), β-actin (A1978, AC-15, Sigma-Aldrich), β-tubulin (T4026, Sigma Aldrich). Antibodies were validated by western blot using the respective recombinant protein as positive control. The following antibodies were used for immunostaining: LC3 (L7543, Sigma), Lysozyme (sc27956, Santa Cruz), Muc2 (ab134119, EPR6145, Abcam), CoxIV (4850, Cell Signaling), PHB1 (70R-5543, Fitzgerald), and Nix (12396, Cell Signaling). Isotype controls were included to validate immunostaining.

Alula K.M., Delgado-Deida Y., Callahan R., Till A., Underwood L., Thompson W.E., Souza R.F., Dassopoulos T., Onyiah J., Venuprasad K, & Theiss A.L. (2023). Inner mitochondrial membrane protein Prohibitin 1 mediates Nix-induced, Parkin-independent mitophagy. Scientific Reports, 13, 18.

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Protein Turnover Assay and Immunoblotting

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Cells were washed twice with cold PBS and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, phosphatase and protease inhibitors). Sample proteins were separated by SDS-PAGE and transferred onto PVDF membranes. For protein turnover assay, cells were treated with 60 μg/mL cycloheximide (MDBio, Inc.) for the indicated times before collection. The following antibodies were used in the immunoblotting and immunoprecipitation experiments: CHD6 (1:500, Abcam, ab114095), CHD6 (1:1000, Santa Cruz, sc-393445), TMEM65 (1:500, Sigma, HPA025020), p-AKT (Ser473) (1:1000, Cell Signaling, 4060S), AKT (1:2000, Cell Signaling, 2920S), Vinculin (1:4000, Cell Signaling, 4650S), FBXW7 (1:5000, Abcam, ab109617), COX IV (1:5000, Cell Signaling, 4850), PPOX (1:2000, Santa Cruz, sc-271768), VDAC1 (1:2000, Santa Cruz, sc-390996), mtTFA (1:1000, Santa Cruz, sc-166965), p-Drp1 (Ser616) (1:800, Cell Signaling, 4494S), Drp1 (1:2000, Proteintech, 12957-1-AP), Parkin (1:1000, Proteintech, 14060-1-AP), GAPDH (1:4000, Proteintech, 60004-1-Ig), Flag-tag (1:5000, Sigma, F1804), HA-tag (1:5000, Cell Signaling, 3724S), Myc-tag (1:5000, Cell Signaling, 2276S), β-Catenin (1:4000, BD, 610153), TCF4 (1:1000, Santa Cruz, sc-166699), GSK3β (1:4000, Cell Signaling, 9832S).

Zhang B., Liu Q., Wen W., Gao H., Wei W., Tang A., Qin B., Lyu H., Meng X., Li K., Jin H., Yu F., Pan Q., Lin J, & Lee M.H. (2022). The chromatin remodeler CHD6 promotes colorectal cancer development by regulating TMEM65-mediated mitochondrial dynamics via EGF and Wnt signaling. Cell Discovery, 8, 130.

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Immunofluorescence Profiling of OXPHOS Complexes

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Immunofluorescence staining was performed on 5-µm sections of frozen tissue, mounted on positively charged slides, as previously described (32 (link)). Staining for complex I (NDUFB8; 1:200, Invitrogen), complex II (SDHA; 1:200, Abcam; Cambridge, UK) and complex IV (COXIV; 1:400, Cell Signaling; Beverly, MA, USA) of the respiratory chain was performed by incubation with the primary antibody at 4°C overnight followed by 1 hour incubation at room temperature with secondary antibodies Alexa Fluor 488 goat anti-rabbit IgG (1:400) and/or Alexa Fluor 594 goat anti-mouse IgG (1:400) (Invitrogen). Slides were counterstained and mounted with Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and analyzed with a fluorescent microscope (Leica DM RXA).

Lang M., Vocke C.D., Merino M.J., Schmidt L.S, & Linehan W.M. (2015). Mitochondrial DNA mutations distinguish bilateral multifocal renal oncocytomas from familial Birt-Hogg-Dubé tumors. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 28(11), 1458-1469.

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Fractionation and Immunoblotting of Cellular Proteins

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Cytosolic and mitochondrial fractions were prepared as described above. Equal amounts of each lysate were fractionated by 4–12% gradient Bis-Tris SDS-PAGE gel electrophoresis. For affinity purified proteins, bead-bound proteins were eluted by boiling for 10 min in elution buffer and size-fractionated by electrophoresis using a 4–12% gradient Bis-Tris SDS-PAGE (Life Technologies). After gel-electrophoresis, the fractionated proteins were electroblotted onto a nitrocellulose-based Transfer Membrane (Life Technologies). Membranes were blocked with ECL Advance Blocking Agent (GE Healthcare) for 1 h, followed by overnight incubation at 4°C with antibodies against KIF5A (ThermoFisher), YB-1 (Millipore), VDAC1 and COXII (Abcam), COXIV, β-actin (Cell Signaling Technology), MAP2 and Tau (Sigma Aldrich) at 1:1000 dilution. Membranes were washed three times with TBS and 0.1% Tween 20 and incubated with HRP-labeled secondary antibody for 1 h at room temperature. After washing, membranes were developed with SuperSignal West Femto Maximum Sensitivity Substrate Detection Kit (Life Technologies).

Vargas J.N., Kar A.N., Kowalak J.A., Gale J.R., Aschrafi A., Chen C.Y., Gioio A.E, & Kaplan B.B. (2016). Axonal localization and mitochondrial association of precursor microRNA 338. Cellular and molecular life sciences : CMLS, 73(22), 4327-4340.

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Autophagy Induction and Analysis

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Rapamycin was purchased from Millipore. H₂O₂ was from Fisher Scientific. 4-hydroxytamoxifen and etoposide was from Sigma-Aldrich. The following antibodies are used: LC3 (MBL #PM036 for WB of MEFs; Cell Signaling Technology #3868 for IP, ChIP, IF, WB; Cell Signaling Technology #2775 for WB), β-tubulin (Sigma-Aldrich #T4026), calreticulin (Cell Signaling Technology #12238), COX IV (Cell Signaling Technology #4850), Atg5 (Cell Signaling Technology #8540), Atg7 (Cell Signaling Technology #8558), Lamin B1 (Abcam #ab16048), Lamin B2 (Abcam #ab8983), Lamins A/C (Millipore #MAB3211), GFP (Roche #11 814 460 001 and Abcam #ab290), p62 (Abnova #H00008878-M01), GAPDH (Fitzgerald Industries #10R-G109A), p16 (Abcam # ab16123), Ras (Millipore #05-516), HA (Sigma-Aldrich #H3663), H3K27me3 (Active Motif # 39538), H3K9me3 (Abcam #ab8898), LAMP1 (Iowa Hybridoma Bank #H4a3-s), and Flag (Sigma-Aldrich #F1804).

Dou Z., Xu C., Donahue G., Shimi T., Pan J.A., Zhu J., Ivanov A., Capell B.C., Drake A.M., Shah P.P., Catanzaro J.M., Ricketts M.D., Lamark T., Adam S.A., Marmorstein R., Zong W.X., Johansen T., Goldman R.D., Adams P.D, & Berger S.L. (2015). Autophagy mediates degradation of nuclear lamina. Nature, 527(7576), 105-109.

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Cellular Protein Extraction and Immunoblotting

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Whole cellular protein extracts were lysed in SDS-extraction buffer. Mitochondrial and cytoplasmic extracts were prepared using a mitochondria isolation kit (ThermoFisher Scientific, Waltham, MA, USA) and Dounce homogenizer. The following primary antibodies were used: DDX3 (1:1000, mAb AO196^{57 (link)}), β-actin (1:10000, A5441, Sigma-Aldrich), OXPHOS complexes (1:1000, ab110411, Abcam, Cambridge UK), COX IV (1:5000, #4850, Cell Signaling Technology, Danvers, MA, USA), PARP (1:1000, #9542, Cell Signaling Technology), Caspase 3 (1:500, #9665, Cell Signaling Technology), and LC3 (1:1000, #1775S, Cell Signaling Technology).

Heerma van Voss M.R., Vesuna F., Bol G.M., Afzal J., Tantravedi S., Bergman Y., Kammers K., Lehar M., Malek R., Ballew M., ter Hoeve N., Abou D., Thorek D., Berlinicke C., Yazdankhah M., Sinha D., Le A., Abrahams R., Tran P.T., van Diest P.J, & Raman V. (2017). Targeting mitochondrial translation by inhibiting DDX3: a novel radiosensitization strategy for cancer treatment. Oncogene, 37(1), 63-74.

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Adipose Tissue Protein Isolation and Western Blot

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Total protein from mouse adipose tissue was prepared in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride) supplemented with a protease inhibitor cocktail (Roche) and phosphatase inhibitors (10 mM NaF, 60 mM β-glycerolphosphate, pH 7.5, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate). Proteins were subjected to SDS-PAGE then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were probed with following antibodies: UCP1 (Abcam, ab10983), Total OXPHOS Rodent Antibody Cocktail (Abcam, ab110413), COXIV (Cell Signaling, 4850), VDAC (Cell Signaling, 4661), HSP90 (Cell Signaling, 4874), phospho-PKA substrate^S/T (Cell Signaling, 9621), phospho-CREB^S133 (Cell Signaling, 9198), CREB (Cell Signaling, 9197), β-actin (Cell Signaling, 8457) and α-tubulin (Cell Signaling, 2144).

Jun H., Ma Y., Chen Y., Gong J., Liu S., Wang J., Knights A.J., Qiao X., Emont M.P., Xu X.Z., Kajimura S, & Wu J. (2020). Adrenergic-independent signaling via CHRNA2 regulates beige fat activation. Developmental cell, 54(1), 106-116.e5.

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Cell Fractionation and Mitochondrial Marker

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Cytosolic and mitochondrial fractions were prepared using the cell fractionation kit-standard (#MS861, Mitosciences, Eugene, OR, USA). The coxIV (Cell Signaling Technology, Danvers, MA, USA) antibody was used as a mitochondrial loading control.

Abbas W., Khan K.A., Kumar A., Tripathy M.K., Dichamp I., Keita M., Mahlknecht U., Rohr O, & Herbein G. (2014). Blockade of BFA-mediated apoptosis in macrophages by the HIV-1 Nef protein. Cell Death & Disease, 5(2), e1080-.

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Antibody Dilutions and Reagent Sources

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Antibodies were used at dilutions indicated and procured from the following sources: H1 (1 : 1000) and p38MAPK (1 : 1000) from Santa Cruz (Santa Cruz, CA); AIF (1 : 500) from Chemicon (Billerica, MA); H1.2 (1 : 1000) from Proteintech Europe (Manchester, UK); H3 (1 : 500), H3Ac (1 : 500), and HP1α (1 : 1000) from Upstate Biotechnology (Lake Placid, NY); α-actin (1 : 500) and α-tubulin (1 : 250) from Neomarker (Fremont, CA); and Bak (1 : 1000), Cox-IV (1 : 250), and p-JNK/SAPK (1 : 500) from Cell Signaling Technology (Beverly, MA). The shRNA plasmids to H1.2 and the scrambled control were from Origene Technologies (Rockville, MD). TMRM (tetramethyl rhodamine methyl ester) was obtained from Sigma (St. Louis); Annexin-V AlexaFluor-488 was from Invitrogen (Carlsbad, USA); anacardic acid, leptomycin B, and SP600125 were from Calbiochem (San Diego, CA).

Garg M., Perumalsamy L.R., Shivashankar G.V, & Sarin A. (2014). The Linker Histone H1.2 Is an Intermediate in the Apoptotic Response to Cytokine Deprivation in T-Effectors. International Journal of Cell Biology, 2014, 674753.

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Mitochondrial Sub-fractionation and Purity Verification

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Two techniques were used to sub-fractionate the mitochondria obtained by cell fractionation. First, isolated mitochondria were incubated with 2% digitonin for 20 minutes, and spun at 9,600g for 15 minutes, which yielded the mitoplasts in the pellet, and the outer mitochondrial membrane (OM) together with the inter-membrane space (IMS) in the supernatant [9 (link),16 (link),17 (link)]. Second, mitochondria were incubated with 2% Nonidet P-40 for 15 minutes and spun at 18,000g for 40 minutes to yield a soluble component including the matrix, IMS and an insoluble fraction composed of mitochondrial inner (IM) and outer membranes (OM) [9 (link)]. The purity of each mitochondrial sub-fraction was verified by western blotting using specific antibodies: COX IV (1:1000, rabbit polyclonal antibodies, Cell Signaling, Danvers, MA) for IM, voltage-dependent anion-selective channel protein 1 [VDAC] (1:1000, rabbit polyclonal antibodies, Cell Signaling, Danvers, MA) for OM, and Grp75 (1:500, Rabbit polyclonal antibody, Abcam, Cambridge, MA) for the matrix.

Rashed E., Lizano P., Dai H., Thomas A., Suzuki C.K., Depre C, & Qiu H. (2015). Heat Shock Protein 22 (Hsp22) Regulates Oxidative Phosphorylation upon Its Mitochondrial Translocation with the Inducible Nitric Oxide Synthase in Mammalian Heart. PLoS ONE, 10(3), e0119537.

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