Cells were cultured in a 6-well plate. After treatment, the total protein of cells was obtained and quantified using a BCA assay (Sigma-Aldrich, St Louis, MO, USA). Proteins were separated according to their molecular weights through sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). After successive incubation with primary and rabbit secondary antibody conjugated with HRP (Abcam, Cambridge, UK), membranes were observed using enhanced chemiluminescence (Pierce, Rockford, IL, USA). Primary antibodies used were anti-GPX-1, anti-CAT, anti-SOD-1, anti-SOD-2, anti-iNOS, anti-COX-2, and anti-β-actin (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA).
Anti sod1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-SOD1 is a primary antibody that specifically recognizes the SOD1 (Superoxide Dismutase 1) protein. SOD1 is an important enzyme responsible for the conversion of superoxide radicals to hydrogen peroxide and oxygen, playing a crucial role in cellular antioxidant defense mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Anti sod2, by Cell Signaling Technology (7 mentions)
Anti catalase, by Cell Signaling Technology (6 mentions)
Anti β actin, by Cell Signaling Technology (4 mentions)
Anti ho 1, by Cell Signaling Technology (4 mentions)
Anti gapdh, by Santa Cruz Biotechnology (4 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti caspase 3, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti atf6, by Cell Signaling Technology (2 mentions)
Anti ire1α, by Cell Signaling Technology (2 mentions)
Anti sod3, by Santa Cruz Biotechnology (2 mentions)
Anti noxa, by Cell Signaling Technology (2 mentions)
Anti survivin, by Cell Signaling Technology (2 mentions)
N acetyl l cysteine nac, by Merck Group (2 mentions)
Anti atf4, by Cell Signaling Technology (2 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (2 mentions)
Anti nox4, by Cell Signaling Technology (2 mentions)
Anti grp94, by Cell Signaling Technology (2 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
20 protocols using anti sod1
1
Antioxidant Protein Expression Analysis
2
Western Blot Analysis of Antioxidant Enzymes
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Protein extracts were obtained by adding sample buffer to the cells, which were scraped and lysed by pipetting and boiling. Equal amounts of protein were loaded and separated in a SDS-PAGE; then, proteins were transferred onto a nitrocellulose membrane and incubated with the proper primary antibody. Membranes were blocked using 7.5% milk in TBS/T buffer. Primary antibodies were prepared in TBS/T buffer as follows: anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH, 1:1000) (Santa Cruz Biotechnologies), anti-SOD2 (1:1000) (Cell Signalling, Danvers, MA, USA), anti-SOD1 (1:1000) (Cell Signalling), anti-catalase (1:500) (Cell Signalling), anti-γH2AX (1:1000) (Abcam, Cambridge, UK), anti-H2AX (1:1000) (Upstate, Darmstadt, Germany), anti-His (1:1000) (Cell Signalling), anti-HA (1:1000) (Roche), anti-FLAG M2 (1:2000) (Sigma-Aldrich), and anti-HPV18 E2 (1:1000) (Abcam). Membranes were washed three times with PBS/T and incubated with HRP coupled secondary anti-mouse or rabbit antibodies (Santa Cruz Biotechnologies). Membranes were finally developed using the ChemoLuminiscent Reagent (Merck Millipore, Billerica, MA, USA) accordingly to the manufacturer's instructions. Densitometric analysis was performed using the ImageJ program ver.1.48h3, National Institutes of Health (NIH).
3
Western Blotting Assay for Protein Analysis
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The western blotting assay detected specific proteins described previously [39 (link)]. In brief, cells were treated with OST 1 h before LPS. The proteins were extracted through PRO-PREP™ and separated by 8–12% SDS-PAGE. The proteins were transferred from the gel to the polyvinyl vinylidene fluoride (PVDF) membranes (Millipore Co., Billerica, MA, USA) and blocked with 5% BSA. Then, they were sampled with primary antibodies overnight and incubated with horseradish peroxidase (HRP) secondary conjugated antibody. The antibody detection response was performed using ECL. The antibody images were captured with the ImageQuant™ Las 4000 Mini Biomolecular Imager (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The antibodies used were anti-Nrf2 (ab62352, Abcam), anti-phospho-Nrf2 (Ser40) (PA5-67520, Thermo Fisher, Waltham, MA, USA), anti-HO-1 (#5853, Cell Signaling, Danvers, MA, USA), anti-SOD1 (#2770, Cell Signaling), anti-Catalase (#14097, Cell Signaling), anti-IL1β (MA5-23691, Thermo Fisher), and anti-TNFα (ab183218, Thermo Fisher).
4
Quantifying Antioxidant Proteins in Tissue Extracts
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Tissue extracts (20 μg of proteins) were resolved in 8% or 15% polyacrylamide gels (SDS-PAGE) under denaturing conditions and then transferred to 0.45 µm polyvinylidene difluoride (PVDF) membranes (Millipore) as per the manufacturer's instructions. The membranes were incubated with the relevant primary antibodies anti-Catalase, anti-SOD1, anti-Hif1α (Cell Signaling Technology, Danvers, MA, USA), anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-VEGF, anti-angiopoietin-2 (Abcam, Cambridge, MA, USA), anti-SOD2 (Merck Millipore, Darmstadt, Germany). The bands were visualized using anti-IgG HRP-conjugated secondary antibodies and ECL Western blotting detection system (Amersham Biosciences, UK) and signal acquisition and quantification were performed using iBright Imager and iBright Analysis Software (Thermo Fisher Scientific, Waltham, Massachusetts, USA).
5
Analyzing Antiviral Protein Expression
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Cultured epithelial cells after stimulation were rinsed with DPBS and proteins were extracted using radio-immunoprecipitation assay (RIPA) buffer (GenDEPOT). Denatured proteins (25 μg) were fractionated on SDS-PAGE and the gels were transferred onto a polyvinylidene difluoride membrane (Bio-Rad, MA, USA). Thereafter the membranes were rinsed and probed with the primary antibodies in the refrigerator overnight at 4 °C; the primary antibodies employed for Western blots were anti-β actin (1:5000) which was obtained from Santa Cruz, USA, anti-viperin, anti-OAS, anti-Mx, anti-TLR3, anti-RIG1, anti-MDA5, anti-SOD1, anti-SOD2, anti-IRF3, and anti-phospho-IRF3 which were obtained from Cell Signaling Technology and used at a dilution of 1:1000.
6
Antioxidant Effects of Korean Red Ginseng
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Korean Red Ginseng powder was kindly provided by the Korea Ginseng Corporation (Daejeon, Korea). The KRG powders were dissolved in distilled water. N-acetyl-l -cysteine (NAC) and Anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-caspase-3, anti-caspase-9, anti-cleaved PARP, anti-BIM, anti-Noxa, anti-survivin, anti-IRE1α, anti-phospho IRE1α, anti-GRP94, anti-eIF2α, anti-phospho eIF2α, anti-ATF6, anti-PERK, anti-phospho PERK, anti-Bip anti-XBP1s, anti-ATF4, anti-SOD2, anti-SOD1, anti-catalase, anti-NOX4, and anti-NOX2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Bak, anti-BAX, anti-Bcl-2, anti-SOD3, and anti-CHOP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-mouse and Rabbit IgG HRP, the secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA).
7
Investigating Antioxidant Proteins in TiO2NP Exposure
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Western blotting analysis was used in order to investigate SOD1 (copper [Cu]/zinc [Zn] SOD), SOD2 (MnSOD), and SIR3 according to previously described protocol.17 (link) Western blotting was used in order to investigate the SOD1, SOD2, and SIR3. Briefly, hFOB 1.19 cells were cultured in 10 cm petri dishes until they reached about 90% confluence and treated with TiO2NPs under SF conditions at the concentrations 25, 50, and 100 μg/mL for 48 hours. Afterwards, conditioned media were discharged and attached cells rinsed with PBS, detached, and homogenized. Following electrophoresis, proteins were transferred onto nitrocellulose membrane (Protran®, Schleicher and Schuell BioScience GmbH, Dassel, Germany) and detected using antibodies: anti-Sirt3, anti-SOD1, and anti-SOD2 antibodies (Cell Signaling Technology, Inc, Danvers, MA, USA). Protein bands were quantified using densitometry software (Bio-Rad Laboratories Inc, Hercules, CA, USA), and normalized using β-actin (Sigma-Aldrich) as a loading control.
8
Western Blot Analysis of Protein Expression
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Cells (2 × 106) were seeded on Petri dishes and induced as described previously. After the incubation time, the cells were detached, and the protein was isolated with RIPA buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich) and 1 mM PMSF (Sigma-Aldrich). Protein concentration was determined by DirectDetect® (Merck Millipore). Thirty micrograms of proteins were separated on 12% polyacrylamide gel (120 V) and transferred on PVDF membranes with wet transfer (100 V, 400 mA, 70 min). Membranes were then blocked in 5% fat-free milk in TBST buffer prior to overnight incubation in 4 °C in the primary antibodies, anti-Akt (#9272S), anti-phospho-Akt (#4060S), anti-p44–42 (Erk1/2, #4695S), anti-phospho-p44–42 (#4370S), anti-SOD1 (#71G8), anti-cleaved PARP1 (#5625, Cell Signaling Technology), and anti-GAPDH (1:1000, sc-59,540, SantaCruz Biotechnology), as a reference. After incubation, the membranes were washed three times with TBST buffer and incubated with secondary antibodies (1:15000, Sigma-Aldrich) for 4 hours at 4 °C. The membranes were washed once again, and the bands were visualized by using Novex® AP Chromogenic Substrate (BCIP/NBT) (Thermo Fisher Scientific). Densitometric analysis was conducted with ImageJ. The experiment was conducted in triplicate.
9
Western Blot Analysis of Cellular Proteins
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The cells (1 × 106) were seeded on Petri dishes, incubated to reach 90% confluency and induced as described previously. Protein isolation, electrophoresis, transfer and Western blot procedure were conducted as described previously [12 (link)]. Primary antibodies were used, i.e., anti-Akt (1:1000 in 5% BSA, Cell Signaling), anti-p44/42 (1:1000 in 5% BSA, Cell Signaling, Danvers, MA, USA), anti-SOD-1 (1:200, Cell Signaling, Danvers, MA, USA) or anti-GAPDH (1:1000, SantaCruz Biotechnology, Dallas, TX, USA), as a reference. Bands were visualized with the Novex® AP Chromogenic Substrate (BCIP/NBT) (Thermo Fisher Scientific, Waltham, MA, USA). A densitometric analysis was conducted in ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). The experiment was conducted in triplicate.
10
Antioxidant and Cytoprotective Assays
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2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH), phenazine methosulfate (PMS), neocaprione, 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), phosphate buffered saline (PBS, pH 7.4), and dimethylsulfoxide (DMSO) were purchased from Sigma Aldrich (St. Louis, MO, USA). DMEM (Dulbecco’s modified Eagle’s medium), fetal bovine serum (FBS), penicillin-streptomycin mixture, 0.25% trypsin-EDTA, were purchased from Gibco-BRL Life Technologies (Grand Island, NY, USA). Antibodies purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) included anti-SOD1, anti-HO-1, anti-catalase (CAT), anti-Glutathione peroxidase 1 (GPx-1), anti-Nrf2, whereas anti-phospho-JNK, Anti-p44/42MAPK (ERK1/2), and anti-phospho-p44/42MAPK (ERK1/2), anti-phospho-p38, Anti-p38 were purchased from Cell Signaling Technology (Beverly, MA, USA).
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