Anti nqo1

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-NQO1 is a laboratory reagent that can be used to detect the presence and quantity of the NQO1 protein in biological samples. NQO1 is an enzyme involved in cellular oxidative stress response. The Anti-NQO1 product provides a specific and reliable method to measure NQO1 levels, which can be useful in various research applications.

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Lab products found in correlation

Close spelling variants of this product

Anti-NAD(P)H:quinone oxidoreductase 1 (NQO1; (2 citations) Anti-NQO1 (A180) (2 citations) Anti-NQO1 (SC-271116) (3 citations) Anti-NQO1 antibody (4 citations) Rabbit anti-NQO1 (3 citations)

40 protocols using anti nqo1

NQO1 and CPT1A Interactome Analysis

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Co-IP was performed with anti-NQO1 (1:1000, Santa Cruz, Heidelberg, Germany) and anti-CPT1A (1:1,000; Abcam, Cambridge, UK) antibodies. Cells were lysed with IP lysis buffer (Beyotime, China). The supernatant, which was collected from centrifugation, was incubated with the anti-NQO1 and anti-CPT1A antibody respectively, overnight at 4°C followed by protein A/G agarose beads (Santa Cruz Biotechnology) for 6 h. Then washed beads with the cold lysis buffer, mixed with 2× SDS buffer (Santa Cruz Biotechnology), and heated denaturation, followed by western blotting analysis.
For the ubiquitination experiment, cells were treated with or without MG132 (10 μM; ApexBio, Houston, USA) for 6 h before being harvested for immunoprecipitation and western blot analysis using anti-Ubiquitin antibody (Santa Cruz Biotechnology).

Xu R., Liu Y., Ma L., Sun Y., Liu H., Yang Y., Jin T, & Yang D. (2023). NQO1/CPT1A promotes the progression of pancreatic adenocarcinoma via fatty acid oxidation: NQO1/CPT1A promotes PAAD via FAO. Acta Biochimica et Biophysica Sinica, 55(5), 758-768.

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Protein Interaction Analysis by Co-IP

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Preparation of cell extracts, co-immunoprecipitation and immunoblotting were carried out as previously described (Pyronnet et al., 1999 (link)). The antibodies used were as follows: anti-eIF4GI and anti-eIF4GII (gifts of Prof. Nahum Sonenberg); anti-DAP5 (CliniSciences #610742); anti-HA-7 (Sigma); anti-β-tubulin (GeneTex #6288022); anti-4E-BP1, anti-NRF2 and anti-p53 (Cell Signaling Technologies #9452, #12721, and #1C12, respectively); anti-Core 20S (Enzo Life Sciences #PW8155); and anti-NQO1 (Santa Cruz #C19).

Alard A., Marboeuf C., Fabre B., Jean C., Martineau Y., Lopez F., Vende P., Poncet D., Schneider R.J., Bousquet C, & Pyronnet S. (2019). Differential Regulation of the Three Eukaryotic mRNA Translation Initiation Factor (eIF) 4Gs by the Proteasome. Frontiers in Genetics, 10, 254.

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Anti-inflammatory and Antioxidant Effects

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GC (PubChem CID: 161120) and PQ were obtained from Sigma-Aldrich (Sigma, MO, USA). Anti-Nrf2, anti-HO-1, anti-NQO-1, anti-GCLM, anti-ICAM-1, anti-VCAM-1, anti-iNOS, anti-IKK-β, anti-IκB-α, anti-NF-κB p65, anti-phosphorylated (p)-IκB-α, anti-Bcl-2, anti-Bcl-xl, anti-Bax, anti-Caspase-3, anti-Caspase-9, anti-GAPDH, anti-β-actin, anti-histone, and IgG-HRP antibodies were products of Santa Cruz Biotechnology (Santa Cruz, Texas, USA). BCA protein concentration assay kit, PVDF membranes, and SDS-PAGE gel preparation kit were purchased from Beyotime Institute of Biotechnology. ECL plus kit was obtained from Nanjing KeyGen Biotech Co., Ltd. (KeyGen, Nanjing, CN). TNF-α, IL-1β, and IL-6 ELISA kits were obtained from Abcam (Cambrige, UK). GSH, NADPH, SOD, CAT, MDA, CK, and LDH kits were products of Nanjing Jiancheng Engineering Institute (Nanjing, CN).

Zhang R., Zhao C., Gong X., Yang J., Zhang G, & Zhang W. (2022). Ginkgolide C Alleviates Acute Lung Injury Caused by Paraquat Poisoning via Regulating the Nrf2 and NF-κB Signaling Pathways. Oxidative Medicine and Cellular Longevity, 2022, 7832983.

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Estrogen-Mediated Regulation of Detoxification Enzymes

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MCF-10A cells were treated with E₂ (1 μM) in the presence and absence of SERMs (DMA, FDMA, Ral; 1 μM). Protein expression of CYP1B1 and 1A1 was analyzed using western blot experiments as previously described (27 (link)). Anti-CYP450 1B1 (Sigma; AV51761), Anti-CYP450 1A1 (Santa Cruz; sc-20772) and anti-β- actin (Cell signaling; #4967) antibodies were used as primary antibodies. Detoxification enzymes were also analyzed using anti-SULT1 (Santa Cruz,CA; sc-32928), anti-SULT1E1 (Santa Cruz, CA; sc-376009), anti-SULT1A1 (Santa Cruz, CA; sc-130883), anti-GSTpi (Cell signaling; #3369), anti-NQO1 (Santa Cruz; sc-32793), and anti COMT (Santa Cruz, CA; sc-25844) as primary antibodies. Antibodies were diluted in blocking solution (5% non fat milk in TBS with 0.1% tween 20). Blots were incubated with primary antibody overnight at 4 °C and with secondary antibody for 1 h at room temperature. Blots were visualized using chemiluminescence substrate (Thermo scientific, Rockford, IL). Imaging and analysis was done using FluroChem software (Cell Biosciences, Santa Clara, CA). Each protein band density was normalized to the respective β- actin band density and was represented as the relative protein expression. Three independent experiments were performed and results were represented as average ± SD.

Madhubhani L.P., Hemachandra P., Patel H., Esala R., Chandrasena P., Choi J., Piyankarage S.C., Wang S., Wang Y., Thayer E., Scism R., Michalsen B.T., Xiong R., Siklos M., Bolton J.L, & Thatcher G.R. (2014). SERMs attenuate estrogen-induced malignant transformation of human mammary epithelial cells by upregulating detoxification of oxidative metabolites. Cancer prevention research (Philadelphia, Pa.), 7(5), 505-515.

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Molecular Signaling Pathway Analysis

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Sodium arsenite and t-RA were purchased from Sigma (St. Louis, MO). Antibodies targeting pERK1/2, total ERK1/2, RARα, RARγ and RXRα were purchased from Cell Signaling Technology (Danvers, MA). Anti-MUC5B, anti-γ-GCSM, anti-HO-1, anti-NQO1, anti-pEGFR (Y1173), anti-EGFR, β-ACTIN, GAPDH and SAM68 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MUC5AC was obtained from Thermo Fisher Scientific (Grand Island, NY). Anti-LPS-free Poly IC (dsRNA) was from InvivoGen (San Diego, CA). IL1β and IL17 was from R&D Systems (Minneapolis, MN).

Liu Y., Liu F., Liang W., Zhu L., Lantz R.C., Zhu J, & Chen Y. (2020). Arsenic represses airway epithelial mucin expression by affecting retinoic acid signaling pathway. Toxicology and applied pharmacology, 394, 114959.

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Evaluating Cellular Oxidative Stress Markers

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IEC-6 cells were plated into 96-well plates (1 × 10⁴ cells/well) and treated as previously indicated for 24 h in order to evaluate COX-2 and iNOS expression and nitrotyrosine formation in IEC-6 cells. In another set of experiments, the IEC-6 cells were incubated with the extract and H₂O₂ as previously indicated, in order to measure HO-1 and NQO1 expression. For this analysis, IEC-6 cells were then collected and washed with phosphate buffered saline (PBS). Fixing solution was added to cells for 20 min and then IEC-6 cells were incubated in fix perm solution for a further 30 min. Anti-COX-2 (BD Transduction Laboratories, Milan, Italy), anti-iNOS (BD Transduction Laboratories, Milan, Italy), anti-nitrotyrosine (Merck Millipore, Milan, Italy), anti-HO-1 (Santa Cruz Biotechnologies, Dallas, TX, USA), or anti-NQO1 (Santa Cruz Biotechnologies, Dallas, TX, USA) antibodies were then added for 1 h. The secondary antibody, in fixing solution, was added to IEC-6 cells and cell fluorescence was then evaluated by a fluorescence-activated cell sorter (FACSscan; Becton Dickinson, Milan, Italy) and analyzed by Cell Quest software (version 4; Becton Dickinson, Milan, Italy), as formerly reported [67 (link)].

Adesso S., Russo R., Quaroni A., Autore G, & Marzocco S. (2018). Astragalus membranaceus Extract Attenuates Inflammation and Oxidative Stress in Intestinal Epithelial Cells via NF-κB Activation and Nrf2 Response. International Journal of Molecular Sciences, 19(3), 800.

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Autophagy and Oxidative Stress Analysis

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Cell lysis and western blot were performed as described previously (Huang M, Leukemia 2013). Antibodies used for Immunoblots: anti-LC3B ( #2775, Cell Signaling), anti-Keap1 (sc-365626, G-2, Santa Cruz Biotechnology), anti-Atg3 (sc-393660, A-3, Santa Cruz Biotechnology), anti-NQO1 (sc-393736, F-8, Santa Cruz Biotechnology), anti-GCLM (Ab124827, Abcam), anti-Bcl2 (sc-56015, 100/D5, Santa Cruz Biotechnology), anti- p62 (sc-25575, H-290, Santa Cruz Biotechnology), anti-actin (A5441, Sigma), and anti-Nrf2 (sc-13032, H-300, Santa Cruz Biotechnology), anti-Brd4 (A301-985A50, BETHYL Laboratories). Anti-CEBPβ (sc-7962, H-7, Santa Cruz Biotechnology).

Huang M., Zhu L., Garcia J.S., Li M.X., Gentles A.J, & Mitchell B.S. (2018). Brd4 regulates the expression of essential autophagy genes and Keap1 in AML cells. Oncotarget, 9(14), 11665-11676.

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Immunoblotting for Nrf2 Pathway Proteins

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Anti-Nrf2, anti-HO-1, anti-β-actin, anti-NQO1, anti-GCLM, and anti-Keap1 were purchased commercially from Santa Cruz. To detect protein expression in total cell lysates, cells were lysed in sample buffer (50 mM Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate (SDS), 10% glycerol, 100 mM dithiothreitol (DTT), 0.1% bromophenol blue) and subjected to immunblotting.

Wu T., Harder B.G., Wong P.K., Lang J.E, & Zhang D.D. (2014). Oxidative Stress, Mammospheres and Nrf2 – New Implication for Breast Cancer Therapy?. Molecular carcinogenesis, 54(11), 1494-1502.

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Western Blot Analysis of Liver Protein Markers

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Western blot analysis was performed in cytosolic and nuclear extracts prepared from liver homogenates. The supernatant fraction was collected and stored at -80°C in aliquots until use. Protein concentration was measured by the Bradford assay [20 (link)]. Lysate proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) for 1 h at room temperature and probed overnight at 4°C with polyclonal anti-NQO1, anti-Keap1, anti-Nrf2, anti-ATF6, and anti BIP/GRP78 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1 : 200-1 : 1.000 dilution with TTBS in 5% nonfat dry milk. Bound primary antibody was detected (HRP—with anti-mouse IgG, anti-rabbit IgG, or anti-goat IgG antibodies) (Sigma-Aldrich, St. Louis, MO, USA). Protein detection was performed via chemiluminescence using a commercial ECL kit (Amersham Pharmacia Biotech, Little Chalfont, Great Britain) [23 (link)]. The density of the specific bands was quantified with imaging densitometer software (Scion Image, Maryland, MA).

Rodrigues G., Moreira A.J., Bona S., Schemitt E., Marroni C.A., Di Naso F.C., Dias A.S., Pires T.R., Picada J.N, & Marroni N.P. (2019). Simvastatin Reduces Hepatic Oxidative Stress and Endoplasmic Reticulum Stress in Nonalcoholic Steatohepatitis Experimental Model. Oxidative Medicine and Cellular Longevity, 2019, 3201873.

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Protein Expression Analysis Workflow

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The treated cells were harvested using radioimmunoprecipitation assay (RIPA) buffer supplemented with a protein inhibitor cocktail (Sigma, St. Louis, MO). The protein concentrations of the cleared lysates were determined using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL), and 20 mg of the total protein was resolved by 4–15% SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA). After electrophoresis, the proteins were electro-transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). The PVDF membrane was then blocked with 5% BSA in phosphate-buffered saline-0.1% Tween 20 (PBST) and then sequentially incubated with specific primary antibodies and HRP-conjugated secondary antibodies. The blots were visualized with the Super Signal enhanced chemiluminescence (ECL) detection system and documented using the Gel Documentation 2000 system (Bio-Rad, Hercules, CA). The antibodies were purchased from the following sources: anti-NRF2 from Epitomics Inc. (Burlingame, CA); anti-NQO1, anti- HO1 and anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA); anti-DNTM1, anti-DNMT3a and anti-DNMT3b from IMGENEX (San Diego, CA); and anti-HDAC 1–6 from Cell Signaling Technology (Danvers, MA).

Khor T.O., Fuentes F., Shu L., Paredes-Gonzalez X., Yang A.Y., Liu Y., Smiraglia D.J., Yegnasubramanian S., Nelson W.G, & Kong A.N. (2014). Epigenetic DNA Methylation of Anti-oxidative Stress Regulator NRF2 in Human Prostate Cancer. Cancer prevention research (Philadelphia, Pa.), 7(12), 1186-1197.

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