10–15 μg proteins obtained from the cell lysates using the RIPA buffer were incubated with the SDS sample buffer (10% glycerol [v/v], Tris-Cl pH 6.8, 2% SDS [w/v], 1% β-mercaptoethanol [v/v], and bromophenol blue) in a heating block at 37 °C for 5 min. After heating, protein samples were applied to western blot analysis. The anti-OXPHOS (1:8000; Abcam) antibody was exploited. The OXPHOS antibody distinguishes 20 kDa NDUFB8 (complex I; NADH: ubiquinone oxidoreductase subunit B8), 30 kDa succinate dehydrogenase complex iron-sulfur subunit B (complex II), 48 kDa UQCRC2 (complex III; ubiquinol-cytochrome c reductase core protein 2), 40 kDa MTCO1 (complex IV; cytochrome c oxidase subunit I), and 55 kDa ATP5A (complex V; ATP synthase).
Anti oxphos
Manufactured by Abcam
Sourced in United States
Anti-OXPHOS is a laboratory product designed to detect and analyze oxidative phosphorylation (OXPHOS) components in various biological samples. It serves as a tool for researchers to investigate the function and expression of the OXPHOS system, which is crucial for cellular energy production.
Automatically generated - may contain errors
Lab products found in correlation
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Anti lc3b, by Cell Signaling Technology (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti ucp1, by Abcam (2 mentions)
Anti vdac1, by Cell Signaling Technology (2 mentions)
Cocktail of protease inhibitor, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Anti tom20, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti pgc 1α, by Cell Signaling Technology (1 mentions)
Anti pink1, by Cell Signaling Technology (1 mentions)
Fotodyne foto analyst luminary workstation systems, by Thermo Fisher Scientific (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Anti mfn2, by Abcam (1 mentions)
Anti micu2, by Merck Group (1 mentions)
Anti grp75, by Santa Cruz Biotechnology (1 mentions)
Anti ip3r1, by Santa Cruz Biotechnology (1 mentions)
Anti pdh e1α, by Abcam (1 mentions)
14 protocols using anti oxphos
1
Protein Isolation and Western Blot Analysis
2
Protein analysis of muscle tissues
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For the protein extracts, myotubes or TA muscles were homogenized in a radioimmunoprecipitation assay (RIPA) buffer with 1 mM of a cocktail of protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) and 1 mM of phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA). Proteins were subjected to SDS-PAGE, transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Waltham, MA, USA), and probed with anti-OXPHOS (1:1000; Abcam, Cambridge, MA, USA), anti-PGC-1α (1:1000, Cell Signaling, Danvers, MA, USA), anti-PINK-1 (1:1000, Cell Signaling, Danvers, MA, USA), anti-LC3B (1:1000, Cell Signaling, Danvers, MA, USA), anti-TOM20 (1:1000, Cell Signaling, Danvers, MA, USA), anti-GAPDH (1:2000; Santa Cruz, Dallas, TX, USA) and anti-β-actin (1:1000; Abcam, Cambridge, MA, USA) antibodies. All immunoreactions were visualized by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA). Images were acquired using the Fotodyne FOTO/Analyst Luminary Workstation Systems (Fisher Scientific, St. Waltham, MA, USA). Densitometry analysis was determined by scanning immunoreactive bands. Intensity values were obtained for further normalization against the control group using ImageJ software (National Institutes of Health [NIH], Bethesda, MD, USA).
3
Immunoblotting Analysis of Mitochondrial Proteins
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Twenty to thirty micrograms of total or mitochondrial protein was separated on SDS-polyacrylamide gel and electro-blotted onto a nitrocellulose membrane (Bio-Rad). Membranes were saturated with blocking buffer for 1 h at room temperature and incubated overnight at 4°C with monoclonal mouse anti-VDAC (1:1000, Abcam), anti-OXPHOS (1:5000, Abcam), anti-IP3R1 (1:1000, Santa Cruz), anti-NDUFA13 (1:1000, Abcam), anti-GAPDH (1:10 000, Abcam) or with polyclonal rabbit anti-MCU (1:500, Abcam), anti-MICU1 (1:500, Thermo Fisher), anti-MICU2 (1:500, Sigma Aldrich), anti-PDH-E1α (1:1000, Abcam), anti-PDHE1α phosphor Ser 293 (1:1000, Abcam), anti-PDHE1α phosphor Ser 300 (1:1000, Millipore), anti-PDHE1α phosphor Ser 232 (1:1000, Calbiochem), anti-PDK4 (1:1000 Novus Biotech), anti-GRP75 (1:1000, Santa Cruz), anti-SIGMA1R (1:1000, Cell Signaling), anti-MFN2 (1:1000, Abcam), and anti-Hsp60 (1:1000, Abcam). Hsp60 and GAPDH were used as loading controls. All immunoblots were developed and quantified using the Odyssey infrared imaging system (LICOR Biosystems) and infrared-labeled secondary antibodies. Band intensities were quantified with ImageJ.
4
Indirubin Screening and Compound Characterization
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The chemical compounds derived from CMAP screening were purchased from Chemical Biology Technology Platform of Chinese Academy of Sciences (shanghai, china) and applied in a dose of 10 μM (final concentration) for each drug within cell experiments. Indirubin used in animal treatment was purchased from MedChemExpress (Catalog No.: HY-N117; CAS No.: 479–41-4; Purity: >98%, Monmouth Junction, NJ). Indirubin was first dissolved in dimethyl sulfoxide (DMSO) and stored in the dark at − 20 °C. During our experiments, Indirubin was diluted into corresponding concentration with the Corn oil (Sigma-Aldrich). All the other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise specified. The primary antibodies included anti-UCP1 (Abcam, ab10983 and ab23841), anti-PGC1α (Santa Cruze Biotechnology, sc-13,067), anti-OXPHOS (Abcam, ab110413), anti- phospho-(Ser/Thr) PKA Substrate (Cell Signaling Technology, #9621), anti-phospho-CREB (Cell Signaling Technology, #4276S, San Antonio, TX, USA), anti-VDAC1 (Cell Signaling Technology, #4661), anti-β-Tubulin (Cell Signaling Technology, #2146).
5
Western Blot Analysis of Mitochondrial Proteins
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The western blot was performed as previously described53 (link). Anti-OXPHOS (Abcam Plc, Cambridge, United Kingdom; Cat# ab110413, RRID: AB_2629281), anti-HSP60 (Santa Cruz Biotechnology, CA, USA; Cat# sc-13115, RRID: AB_627758), anti-YME1L1 (Proteintech, Rosemont, Illinois, USA; Cat# 11510-1-AP, RRID: AB_2217459), anti-PINK1 (Proteintech, Rosemont, Illinois, USA; 23274-1-AP), anti-OPA1 (Cell Signaling Technology; Cat# 80471, RRID: AB_2734117), anti-VDAC (Cell Signaling Technology; Cat# 4866, RRID: AB_2272627), anti-DRP1 (Cell Signaling Technology; Cat# 8570, RRID: AB_10950498), anti-α-tubulin (Cell Signaling Technology; Cat# 2144, RRID: AB_2210548) and anti-GAPDH (Cell Signaling Technology; Cat# 2118, RRID: AB_561053), anti-LONP1 (Bioss Antibodies, Boston, Massachusetts, USA; Cat# bs-4245R, RRID: AB_11051909). All antibodies were used at dilution 1:1000. Ponceau was from Sigma-Aldrich (Saint Louis, MO, USA). The respective loading control normalized the western blotting results, and the final values were given in the percentage of the respective control group.
6
Protein Extraction and Western Blot Analysis
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Proteins were extracted from cells in RIPA buffer (Beyotime, #P0013B) with protease and phosphatase inhibitor. Cell lysates were centrifuged at 12,000 g for 30 min at 4°C, and the protein concentration was measured by BCA Protein Assay Kit (EpiZyme, #ZJ102). Proteins were separated by SDS‐PAGE gel and transferred to 0.45 μm PVDF membranes (Millipore, #IPVH00010), which were then blocked with 5% skim milk for 1 h at room temperature. Primary antibodies were incubated with the membrane at 4°C overnight, including anti‐emerin (1:1000 #PA5‐79201) purchased from Invitrogen; anti‐OXPHOS (1:500, #ab110413) purchased from Abcam; anti‐PGC1α (1:1000, #A12348) purchased from ABclonal Technology; anti‐DRP1 (1:1000, #8570), anti‐MFN2 (1:1000, #9482) and anti‐GAPDH (1:2000 #5174) purchased from Cell Signalling Technology. After washing thrice in TBST, membranes were incubated with horseradish peroxidase (HRP)‐labelled Goat Anti‐Rabbit IgG (Beyotime, #A0208) or Goat Anti‐Mouse IgG (Beyotime, #A0216). Following addition of ECL Prime Western Blotting Detection Reagent (Amersham Bioscience, #RPN2232), immunoblot signal was detected by GeneGnome XRQ Chemiluminescence Imaging System (Syngene). Intensity of bands was quantified using ImageJ software.
7
Western Blot Analysis of OXPHOS Proteins
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The protein was extracted from PBMCs using a radioimmunoprecipitation assay (RIPA) buffer. The total protein (0.3 mg/mL) was mixed with loading buffer, loaded onto 12.5% SDS‐acrylamide gels and then transferred to nitrocellulose membranes in a glycine/methanol‐transfer buffer using a Wet/Tank blotting system (Bio‐Rad Laboratories). Membranes were blocked in 5% non‐fat dry milk in Tris‐Buffered saline and Tween buffer for 1 hour, and the membranes were incubated with anti‐OXPHOS (1:500 dilution; Abcam) overnight at 4°C. Actin (1:1000 dilution; Santa Cruz Biotechnology) was used as a housekeeping protein. Bound antibodies were detected using horseradish peroxidase‐conjugated with antimouse IgG (1:500 dilution; Cell Signaling). The membranes were exposed to an ECL Western blotting substrate (Bio‐Rad Laboratories), and the densitometric analysis was carried out using a ChemiDoc Touch Imaging System (Bio‐Rad Laboratories).
8
Cardiac Ventricle Protein Extraction
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Cardiac left ventricle were homogenized in lysis buffer (Tris-HCl 50 mM, pH 7.4 + NaCl 150 mM, EDTA 1 mM, Triton x-100 1%, Sodium deoxycholate 100 mM, SDS 1%, Sodium pyrophosphate 10 mM, NaF 100 mM, Sodium orthovanadate 10 mM and protease inhibitor cocktail) using the Ultra-Turrax T25 basic homogenizer (IKA®) at 9500 L/min. Samples were held on ice for 1 h and subsequently centrifuged at 12000 x g for 15 min at 4 °C. The supernatant was collected for protein dosage with Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer`s instructions. Samples (40 μg of total protein) were resolved in SDS-PAGE 15% and transferred to a Polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After transfer, membrane was blocked with 5% nonfat dry milk for 2 h and incubated with anti-oxphos (Abcam) and anti-vinculin (Sigma-Aldrich), overnight at 4 °C. Then membrane was washed and incubated with HRP-conjugated specific secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Finally, membrane was developed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The capture of chemiluminescent signal was obtained using a Imagequant LAS4000 (GE Healthcare Life Sciences) equipment.
9
Protein Extraction and Western Blot Analysis
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Tissues were lysed in RIPA buffer containing 0.1% SDS, 1% NP-40, 0.5% Na deoxycholate, 150 mM NaCl, 50 mMTris-Cl (pH 8.0), and 1 mMEDTA supplemented with protease inhibitor (Roche). Total proteins were loaded and fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected with the antibodies anti-NFIA (1:500 dilution, Sigma, HPA006111), anti-Ox-Phos (1:1,000 dilution, Abcam, 110413), anti-actin (1:1,000 dilution, Santa Cruz Biotechnology, sc-1616), anti-NF-κB p65 (1:1,000 dilution, Cell Signaling, 8242), and FLAG M2 (1:2,000 dilution, Sigma, F3165).
10
Western Blot Analysis of Cellular Proteins
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Total protein concentrations from cells or tissues were detected using Enhanced BCA Protein Assay Kit (Thermo Scientific). Equivalent amounts of protein were resolved by SDS-PAGE on 10% Tris-glycine gradient gel and transferred to a NC membrane (PALL). Membranes were blocked with 5% nonfat dry milk and incubated overnight at 4 ℃ with the following primary antibodies, including anti-UCP1 (Abcam), anti-PGC1α (Santa Biotechnology, USA), anti-OXPHOS (Abcam) and anti-β-actin (Santa Biotechnology). After washing with PBST (1 L PBS with 500 μl Tween-20) for three times, membranes were incubated for 2 h at RT in the corresponding HRP-conjugated secondary antibodies. After washing in TBST, detection was performed using Odyssey ®CLx Imaging System (LI-COR).
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