anti nqo1 antibody Cell Signaling Technology Inc Search Results


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  • 94
    Cell Signaling Technology Inc nqo1
    The triterpenoid, CDDO-Im, a Nrf2 inducer, reduces zymosan-induced lung inflammation and pro-inflammatory BALF cytokines in p47 phox−/− mice. CDDO-Im (0.2 mg/mouse by i.p. injection) or vehicle (control) was administered daily to p47 phox−/− mice from day −1 to +2 in relation to i.t. zymosan, and BALF and lungs were harvested on day +3. Representative H E stained lung sections of p47 phox−/− mice administered zymosan plus vehicle (A) or zymosan plus CDDO-Im (B). Neutrophil (C) and cytokine (D) concentrations were assessed in BALF obtained at day 3 after zymosan treatment. Significant differences were observed for neutrophils (p = 0.03), IL-23 (p = 0.008), IL-17 (p = 0.02), TNF-α (p = 0.02), and LIX (p = 0.03) (Mann-Whitney two-tailed test). E) Lung NF-κB activation, measured by bioluminescence, was similar in p47 phox−/− /HLL mice administered zymosan plus CDDO-Im versus zymosan plus vehicle (Two-way ANOVA, p = NS). F) Representative Western blot of lung homogenates for <t>NQO1</t> and (G) densitometry (normalized to β-actin) (G) for 3 mice per genotype per treatment (p
    Nqo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti nqo1 a180
    The triterpenoid, CDDO-Im, a Nrf2 inducer, reduces zymosan-induced lung inflammation and pro-inflammatory BALF cytokines in p47 phox−/− mice. CDDO-Im (0.2 mg/mouse by i.p. injection) or vehicle (control) was administered daily to p47 phox−/− mice from day −1 to +2 in relation to i.t. zymosan, and BALF and lungs were harvested on day +3. Representative H E stained lung sections of p47 phox−/− mice administered zymosan plus vehicle (A) or zymosan plus CDDO-Im (B). Neutrophil (C) and cytokine (D) concentrations were assessed in BALF obtained at day 3 after zymosan treatment. Significant differences were observed for neutrophils (p = 0.03), IL-23 (p = 0.008), IL-17 (p = 0.02), TNF-α (p = 0.02), and LIX (p = 0.03) (Mann-Whitney two-tailed test). E) Lung NF-κB activation, measured by bioluminescence, was similar in p47 phox−/− /HLL mice administered zymosan plus CDDO-Im versus zymosan plus vehicle (Two-way ANOVA, p = NS). F) Representative Western blot of lung homogenates for <t>NQO1</t> and (G) densitometry (normalized to β-actin) (G) for 3 mice per genotype per treatment (p
    Anti Nqo1 A180, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc mouse anti nqo1
    KOK up-regulates Keap1-Nrf2 signaling in the SNpc and striatum following MPTP intoxication. (A) At 7 days after the last MPTP intoxication, SNpc and striatal sections ( n = 5 per group) were subjected to MitoSOX staining. The staining intensity was then quantified. Insets display high magnification micrographs of areas marked with squares. (B,C) Seven days after the last MPTP intoxication, SNpc, and striatum sample ( n = 3 per group) were performed by Western blot assay. Keap1, Nrf2, HO-1, and <t>NQO1.</t> SNpc (B) and striatum (C) . The top panels illustrate representative Western blots. ANOVA test; ## p
    Mouse Anti Nqo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc anti nqo1 rabbit monoclonal antibody
    KOK up-regulates Keap1-Nrf2 signaling in the SNpc and striatum following MPTP intoxication. (A) At 7 days after the last MPTP intoxication, SNpc and striatal sections ( n = 5 per group) were subjected to MitoSOX staining. The staining intensity was then quantified. Insets display high magnification micrographs of areas marked with squares. (B,C) Seven days after the last MPTP intoxication, SNpc, and striatum sample ( n = 3 per group) were performed by Western blot assay. Keap1, Nrf2, HO-1, and <t>NQO1.</t> SNpc (B) and striatum (C) . The top panels illustrate representative Western blots. ANOVA test; ## p
    Anti Nqo1 Rabbit Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse polyclonal anti nqo1 antibody
    KOK up-regulates Keap1-Nrf2 signaling in the SNpc and striatum following MPTP intoxication. (A) At 7 days after the last MPTP intoxication, SNpc and striatal sections ( n = 5 per group) were subjected to MitoSOX staining. The staining intensity was then quantified. Insets display high magnification micrographs of areas marked with squares. (B,C) Seven days after the last MPTP intoxication, SNpc, and striatum sample ( n = 3 per group) were performed by Western blot assay. Keap1, Nrf2, HO-1, and <t>NQO1.</t> SNpc (B) and striatum (C) . The top panels illustrate representative Western blots. ANOVA test; ## p
    Mouse Polyclonal Anti Nqo1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti nqo1 3187 antibodies
    KOK up-regulates Keap1-Nrf2 signaling in the SNpc and striatum following MPTP intoxication. (A) At 7 days after the last MPTP intoxication, SNpc and striatal sections ( n = 5 per group) were subjected to MitoSOX staining. The staining intensity was then quantified. Insets display high magnification micrographs of areas marked with squares. (B,C) Seven days after the last MPTP intoxication, SNpc, and striatum sample ( n = 3 per group) were performed by Western blot assay. Keap1, Nrf2, HO-1, and <t>NQO1.</t> SNpc (B) and striatum (C) . The top panels illustrate representative Western blots. ANOVA test; ## p
    Anti Nqo1 3187 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc primary antibodies against nqo1
    <t>NQO1</t> controls mitotic progression. (A) Control (ctr) siRNA and NQO1 siRNA transfected MCF-7 cells were synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h) followed by release in normal medium. Flow cytometric analysis of cell cycle at the indicated time points after release was done using propidium iodide DNA staining. (B) Table shows percentage of cells in each phase of the cell cycle (G0/G1, S, and G2/M) at indicated time points after nocodazole release from the experiment presented in (A). (C) Control shRNA and NQO1 shRNA stable MCF-7 cells were synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h). Cell cycle distribution was assessed as in A. (D) Mitotic progression of MCF-7 cells transfected with either control (ctr) siRNA or NQO1 siRNA was monitored by live-cell microscopy. Characteristic phase-contrast images are shown. Mitosis is completed in less than 90 min under normal conditions (arrow, upper). Mitosis fails to be completed as evidenced by the lack of formation of two daughter cells (arrow, lower). (E) MCF-7 cells stably expressing H2B-GFP were transfected with either control (ctr) siRNA or NQO1 siRNA. Mitosis was monitored by live-cell microscopy. Characteristic images are shown. Normal completion of mitosis is shown in the upper panel, whereas impaired mitoses are shown in the lower panel. Scale bar is 10 µm. (F) Percentage of cells exhibiting mitotic defects under different experimental conditions. Mitoses were checked in control (ctr) siRNA, NQO1 siRNA and NQO1 siRNA treated with Nicotinamide mononucleotide (ΝΜΝ, 0.5 mΜ) MCF-7 cells.
    Primary Antibodies Against Nqo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 83/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc nadph quinone oxidoreductase
    The schematic diagram of the antioxidant molecular mechanisms of matrine‐type alkaloids. AGE s (advanced glycation end products) trigger intracellular ROS (reactive oxygen species) production, which induces apoptosis of aortic endothelial cells. The matrine‐type alkaloids administration promotes the phosphorylation of MKK 3 and MKK 6. As the kinases of <t>p38</t> MAPK , phosphorylated MKK 3 and MKK 6 further phosphorylate p38 MAPK, which facilitate the nuclear translocation of Nrf2. Binding with ARE (antioxidant response element), the nuclear transcription factor Nrf2 initiates the transcriptions of antioxidant proteins such as NQO 1 <t>(NADPH</t> quinone oxidoreductase 1) and HO 1 (heme oxygenase 1). Thus, the intracellular ROS accumulation is alleviated and apoptosis is suppressed.
    Nadph Quinone Oxidoreductase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse anti nicotinamide adenine dinucleotide phosphate dehydrogenase quinone 1
    The schematic diagram of the antioxidant molecular mechanisms of matrine‐type alkaloids. AGE s (advanced glycation end products) trigger intracellular ROS (reactive oxygen species) production, which induces apoptosis of aortic endothelial cells. The matrine‐type alkaloids administration promotes the phosphorylation of MKK 3 and MKK 6. As the kinases of <t>p38</t> MAPK , phosphorylated MKK 3 and MKK 6 further phosphorylate p38 MAPK, which facilitate the nuclear translocation of Nrf2. Binding with ARE (antioxidant response element), the nuclear transcription factor Nrf2 initiates the transcriptions of antioxidant proteins such as NQO 1 <t>(NADPH</t> quinone oxidoreductase 1) and HO 1 (heme oxygenase 1). Thus, the intracellular ROS accumulation is alleviated and apoptosis is suppressed.
    Mouse Anti Nicotinamide Adenine Dinucleotide Phosphate Dehydrogenase Quinone 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 81/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Cell Signaling Technology Inc anti nqo 1 antibody
    The schematic diagram of the antioxidant molecular mechanisms of matrine‐type alkaloids. AGE s (advanced glycation end products) trigger intracellular ROS (reactive oxygen species) production, which induces apoptosis of aortic endothelial cells. The matrine‐type alkaloids administration promotes the phosphorylation of MKK 3 and MKK 6. As the kinases of <t>p38</t> MAPK , phosphorylated MKK 3 and MKK 6 further phosphorylate p38 MAPK, which facilitate the nuclear translocation of Nrf2. Binding with ARE (antioxidant response element), the nuclear transcription factor Nrf2 initiates the transcriptions of antioxidant proteins such as NQO 1 <t>(NADPH</t> quinone oxidoreductase 1) and HO 1 (heme oxygenase 1). Thus, the intracellular ROS accumulation is alleviated and apoptosis is suppressed.
    Anti Nqo 1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 81/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The triterpenoid, CDDO-Im, a Nrf2 inducer, reduces zymosan-induced lung inflammation and pro-inflammatory BALF cytokines in p47 phox−/− mice. CDDO-Im (0.2 mg/mouse by i.p. injection) or vehicle (control) was administered daily to p47 phox−/− mice from day −1 to +2 in relation to i.t. zymosan, and BALF and lungs were harvested on day +3. Representative H E stained lung sections of p47 phox−/− mice administered zymosan plus vehicle (A) or zymosan plus CDDO-Im (B). Neutrophil (C) and cytokine (D) concentrations were assessed in BALF obtained at day 3 after zymosan treatment. Significant differences were observed for neutrophils (p = 0.03), IL-23 (p = 0.008), IL-17 (p = 0.02), TNF-α (p = 0.02), and LIX (p = 0.03) (Mann-Whitney two-tailed test). E) Lung NF-κB activation, measured by bioluminescence, was similar in p47 phox−/− /HLL mice administered zymosan plus CDDO-Im versus zymosan plus vehicle (Two-way ANOVA, p = NS). F) Representative Western blot of lung homogenates for NQO1 and (G) densitometry (normalized to β-actin) (G) for 3 mice per genotype per treatment (p

    Journal: PLoS ONE

    Article Title: NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

    doi: 10.1371/journal.pone.0009631

    Figure Lengend Snippet: The triterpenoid, CDDO-Im, a Nrf2 inducer, reduces zymosan-induced lung inflammation and pro-inflammatory BALF cytokines in p47 phox−/− mice. CDDO-Im (0.2 mg/mouse by i.p. injection) or vehicle (control) was administered daily to p47 phox−/− mice from day −1 to +2 in relation to i.t. zymosan, and BALF and lungs were harvested on day +3. Representative H E stained lung sections of p47 phox−/− mice administered zymosan plus vehicle (A) or zymosan plus CDDO-Im (B). Neutrophil (C) and cytokine (D) concentrations were assessed in BALF obtained at day 3 after zymosan treatment. Significant differences were observed for neutrophils (p = 0.03), IL-23 (p = 0.008), IL-17 (p = 0.02), TNF-α (p = 0.02), and LIX (p = 0.03) (Mann-Whitney two-tailed test). E) Lung NF-κB activation, measured by bioluminescence, was similar in p47 phox−/− /HLL mice administered zymosan plus CDDO-Im versus zymosan plus vehicle (Two-way ANOVA, p = NS). F) Representative Western blot of lung homogenates for NQO1 and (G) densitometry (normalized to β-actin) (G) for 3 mice per genotype per treatment (p

    Article Snippet: Analysis of Nrf2 Activation Western blot analysis of nuclear protein fractions was performed by Odyssey system (LI-COR Bioscience, Nebraska USA), using antibodies specific for Nrf2, TBP, beta-actin (Santa Cruz Technology), NQO1 (Cell Signaling Technology).

    Techniques: Mouse Assay, Injection, Staining, MANN-WHITNEY, Two Tailed Test, Activation Assay, Western Blot

    p62 Ablation Attenuates Pancreatitis-Accelerated Neoplastic Progression (A) Quantification of ADM and PanIN1 density, amylase and Ki67 staining of pancreatic sections of indicated 5-week-old mice (n = 5). (B) H E staining, amylase IHC, amylase and CK19 co-IF, and Sox9 IHC of pancreatic sections from indicated 5-week-old mice. Scale bars: 25 µm. (C) Kaplan-Meier survival curves of indicated mouse strains (n = 10). (D) Q-RT-PCR analysis of mRNA in acinar and ductal cell fractions from indicated 5-week-old mice (n = 5). (E) NQO1, MDM2 and HES1 IHC of pancreatic sections from indicated 5-week-old mice. Scale bars: 25 µm. (F) p53 IB analysis of pancreatic lysates from 5-week-old mice of indicated genotypes. (G) Frequency of ALDH expression in EpCAM + cells from 8-week-old Kras G12D (n = 3), Kras G12D ; Ikkα Δpan (n = 7), and Kras G12D ; Ikkα/p62 Δpan (n = 4) mice. (H) Sphere-forming capacity of isolated ALDH + .

    Journal: Cancer cell

    Article Title: Stress Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas

    doi: 10.1016/j.ccell.2017.10.011

    Figure Lengend Snippet: p62 Ablation Attenuates Pancreatitis-Accelerated Neoplastic Progression (A) Quantification of ADM and PanIN1 density, amylase and Ki67 staining of pancreatic sections of indicated 5-week-old mice (n = 5). (B) H E staining, amylase IHC, amylase and CK19 co-IF, and Sox9 IHC of pancreatic sections from indicated 5-week-old mice. Scale bars: 25 µm. (C) Kaplan-Meier survival curves of indicated mouse strains (n = 10). (D) Q-RT-PCR analysis of mRNA in acinar and ductal cell fractions from indicated 5-week-old mice (n = 5). (E) NQO1, MDM2 and HES1 IHC of pancreatic sections from indicated 5-week-old mice. Scale bars: 25 µm. (F) p53 IB analysis of pancreatic lysates from 5-week-old mice of indicated genotypes. (G) Frequency of ALDH expression in EpCAM + cells from 8-week-old Kras G12D (n = 3), Kras G12D ; Ikkα Δpan (n = 7), and Kras G12D ; Ikkα/p62 Δpan (n = 4) mice. (H) Sphere-forming capacity of isolated ALDH + .

    Article Snippet: The following antibodies were used: α-amylase (Millipore Sigma, A8273), Cytokeratin 19 (Santa Cruz, sc-33111), Ki67 (GeneTex, GTX16667), IKKα (Novus Biologicals, 14A231), p62 (Progen, GP62-C), Nrf2 (Santa Cruz, sc-722), Nqo1 (Cell Signaling, #3187), Nqo1 (Santa Cruz, sc-16464), Sox9 (Santa Cruz, sc-20095), MDM2 (Millipore Sigma OP 115), Hes1 (Santa Cruz, sc-25392), cyclin D1 (Cell Signaling, #2978), αSMA (abcam, ab5694), F4/80 (Caltag Medsystems, PSI-76-052).

    Techniques: Staining, Mouse Assay, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation

    Effects of RTA 408 on expression of Nrf2 and downstream genes . (A) Dose-dependent effects of RTA 408 on Nrf2 and downstream gene expression. RPE cells were pretreated with 1–100 nM RTA for 24 h. Control cells were treated identically, but without addition of RTA 408. Whole cell lysates of each group were prepared, and 60 ug of each protein sample was used to detect the levels of Nrf2, SOD2, catalase, NQO1, HO-1, Grx1, and Trx1 by western immunoblotting. GAPDH was used as a loading control. (B) Time-dependent effects of RTA 408 on Nrf2 and its downstream gene expression. Cells were treated with 100 nM RTA for various time (0–24 h). 60 μg of each protein sample was subjected to western immunoblot analysis. The blot was incubated with GAPDH, Nrf2, SOD2, catalase, NQO1, HO-1, Grx1, and Trx1 antibodies. GAPDH was used as a reference for equal protein loading. All experiments were repeated three times with similar results. (C) Nrf2 expression in the cytoplasmic and nuclear fractions. Cells were treated with 100 nM of RTA 408 for various time points (0, 0.5, 2 and 6 h) and then were separated to its nuclear and cytoplasmic portions using a kit. 40 μg of each sample were subjected to western immunoblot analysis and incubated with B23, Nrf2, and β-actin antibodies. All experiments were repeated three times with similar results. (D) Quantitative analysis of time-dependent Nrf2 translocation are depicted as mean±SD, ** P

    Journal: Redox Biology

    Article Title: The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2) activation

    doi: 10.1016/j.redox.2015.12.005

    Figure Lengend Snippet: Effects of RTA 408 on expression of Nrf2 and downstream genes . (A) Dose-dependent effects of RTA 408 on Nrf2 and downstream gene expression. RPE cells were pretreated with 1–100 nM RTA for 24 h. Control cells were treated identically, but without addition of RTA 408. Whole cell lysates of each group were prepared, and 60 ug of each protein sample was used to detect the levels of Nrf2, SOD2, catalase, NQO1, HO-1, Grx1, and Trx1 by western immunoblotting. GAPDH was used as a loading control. (B) Time-dependent effects of RTA 408 on Nrf2 and its downstream gene expression. Cells were treated with 100 nM RTA for various time (0–24 h). 60 μg of each protein sample was subjected to western immunoblot analysis. The blot was incubated with GAPDH, Nrf2, SOD2, catalase, NQO1, HO-1, Grx1, and Trx1 antibodies. GAPDH was used as a reference for equal protein loading. All experiments were repeated three times with similar results. (C) Nrf2 expression in the cytoplasmic and nuclear fractions. Cells were treated with 100 nM of RTA 408 for various time points (0, 0.5, 2 and 6 h) and then were separated to its nuclear and cytoplasmic portions using a kit. 40 μg of each sample were subjected to western immunoblot analysis and incubated with B23, Nrf2, and β-actin antibodies. All experiments were repeated three times with similar results. (D) Quantitative analysis of time-dependent Nrf2 translocation are depicted as mean±SD, ** P

    Article Snippet: Antibodies listed below were used: anti-Nrf2 (Cell Signaling, Beverly, MA, USA, #12721), anti-NQO1 (Cell Signaling, #3187), anti-Bax (Cell Signaling, #2772), anti-Bcl2 (Cell Signaling, #2876), anti-cleaved caspase 3 (17 kDa) (Cell Signaling, #9664), anti-HO-1 (Cell Signaling, #5061), anti-Grx1 (Abcam, Cambridge, MA, USA, ab45953), anti-Trx1 (Abcam, ab86255), anti-PSSG (Virogen, Watertown, MA, USA, 101-A-100), anti-SOD2 (signal, HPA001814), anti-catalase (Abcam, ab16731), anti-GAPDH (Santa Cruz, Santa Cruz, CA, USA, sc-32233), anti-B23 antibodies, and horseradish peroxidase-conjugated secondary antibodies (sc2061, sc2060, sc2030; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Expressing, Western Blot, Incubation, Translocation Assay

    Effects of RTA 408 on Grx1, Trx1, and NQO1 enzyme activities . RPE cells were pretreated with 100 nM RTA 408 for 24 h followed by 200 μM H 2 O 2 for another 6 h. Quantitative analysis of Grx1 (A), Trx1 (B), and (C) NQO1 activity of all treatment groups (control, H 2 O 2 -treated, RTA 408 and H 2 O 2 -treated, and 100 nM RTA 408 only RPE cells) are depicted as mean±SD, ** P

    Journal: Redox Biology

    Article Title: The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2) activation

    doi: 10.1016/j.redox.2015.12.005

    Figure Lengend Snippet: Effects of RTA 408 on Grx1, Trx1, and NQO1 enzyme activities . RPE cells were pretreated with 100 nM RTA 408 for 24 h followed by 200 μM H 2 O 2 for another 6 h. Quantitative analysis of Grx1 (A), Trx1 (B), and (C) NQO1 activity of all treatment groups (control, H 2 O 2 -treated, RTA 408 and H 2 O 2 -treated, and 100 nM RTA 408 only RPE cells) are depicted as mean±SD, ** P

    Article Snippet: Antibodies listed below were used: anti-Nrf2 (Cell Signaling, Beverly, MA, USA, #12721), anti-NQO1 (Cell Signaling, #3187), anti-Bax (Cell Signaling, #2772), anti-Bcl2 (Cell Signaling, #2876), anti-cleaved caspase 3 (17 kDa) (Cell Signaling, #9664), anti-HO-1 (Cell Signaling, #5061), anti-Grx1 (Abcam, Cambridge, MA, USA, ab45953), anti-Trx1 (Abcam, ab86255), anti-PSSG (Virogen, Watertown, MA, USA, 101-A-100), anti-SOD2 (signal, HPA001814), anti-catalase (Abcam, ab16731), anti-GAPDH (Santa Cruz, Santa Cruz, CA, USA, sc-32233), anti-B23 antibodies, and horseradish peroxidase-conjugated secondary antibodies (sc2061, sc2060, sc2030; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Activity Assay

    RTA 408 activates the Nrf2 pathway which leads to an upregulation of antioxidant enzymes and protects the cell from oxidative stress . Binding of RTA 408 to Cys151 in Keap1, the negative regulator of Nrf2, results in Keap1 inhibition. This promotes Nrf2 movement into the nucleus where it binds to the antioxidant response element (ARE). With the activation of ARE, transcriptional activation of antioxidant enzymes heme oxygenase-1 (HO-1), NADPH dehydrogenase (NQO1), superoxide dismutase 2 (SOD2), catalase, thioredoxin 1 (Trx1), and glutaredoxin 1 (Grx1) occurs. HO-1 converts heme to carbon monoxide, iron (II), and biliverdin, which all indirectly scavenge ROS. NQO1 converts enzymes and other proteins (R) back to their reduced form (RH) through the electron transfer between NADPH and NADP. Superoxide (O 2 − ) can be converted to hydrogen peroxide using SOD2 and then further processed into water by catalase. Grx1 and Trx1 work together to reduce protein-glutathione mixed disulfide (PSSG) and protein-protein disulfide (PSSP) to protect protein thiols from oxidation. Overall, RTA 408 induction of phase II antioxidant enzymes such as HO-1, NQO1, SOD2, catalase, Grx1, and Trx1 via activation of Nrf2 promote RPE cell survival during oxidative stress.

    Journal: Redox Biology

    Article Title: The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2) activation

    doi: 10.1016/j.redox.2015.12.005

    Figure Lengend Snippet: RTA 408 activates the Nrf2 pathway which leads to an upregulation of antioxidant enzymes and protects the cell from oxidative stress . Binding of RTA 408 to Cys151 in Keap1, the negative regulator of Nrf2, results in Keap1 inhibition. This promotes Nrf2 movement into the nucleus where it binds to the antioxidant response element (ARE). With the activation of ARE, transcriptional activation of antioxidant enzymes heme oxygenase-1 (HO-1), NADPH dehydrogenase (NQO1), superoxide dismutase 2 (SOD2), catalase, thioredoxin 1 (Trx1), and glutaredoxin 1 (Grx1) occurs. HO-1 converts heme to carbon monoxide, iron (II), and biliverdin, which all indirectly scavenge ROS. NQO1 converts enzymes and other proteins (R) back to their reduced form (RH) through the electron transfer between NADPH and NADP. Superoxide (O 2 − ) can be converted to hydrogen peroxide using SOD2 and then further processed into water by catalase. Grx1 and Trx1 work together to reduce protein-glutathione mixed disulfide (PSSG) and protein-protein disulfide (PSSP) to protect protein thiols from oxidation. Overall, RTA 408 induction of phase II antioxidant enzymes such as HO-1, NQO1, SOD2, catalase, Grx1, and Trx1 via activation of Nrf2 promote RPE cell survival during oxidative stress.

    Article Snippet: Antibodies listed below were used: anti-Nrf2 (Cell Signaling, Beverly, MA, USA, #12721), anti-NQO1 (Cell Signaling, #3187), anti-Bax (Cell Signaling, #2772), anti-Bcl2 (Cell Signaling, #2876), anti-cleaved caspase 3 (17 kDa) (Cell Signaling, #9664), anti-HO-1 (Cell Signaling, #5061), anti-Grx1 (Abcam, Cambridge, MA, USA, ab45953), anti-Trx1 (Abcam, ab86255), anti-PSSG (Virogen, Watertown, MA, USA, 101-A-100), anti-SOD2 (signal, HPA001814), anti-catalase (Abcam, ab16731), anti-GAPDH (Santa Cruz, Santa Cruz, CA, USA, sc-32233), anti-B23 antibodies, and horseradish peroxidase-conjugated secondary antibodies (sc2061, sc2060, sc2030; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Binding Assay, Inhibition, Activation Assay

    The effect of ZJF pretreatment on the nuclear factor kappa B p65 (NF-κB p65), nuclear factor erythroid 2-related factor 2 (Nrf-2), and NAD(P)H: quinone oxidoreductase 1 (NQO1 ) protein expression in response to the APAP-induced hepatotoxicity. ( A ) Western blotting analyses of NF-κB p65, Nrf-2, and NQO1 proteins; quantitative densitometric analyses of ( B ) NF-κB p65, ( C ) Nrf-2, and ( D ) NQO1 proteins normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each value represents the mean ± SEM of three independent experiments. ## p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Protective Effect of Flavonoids from Ziziphus jujuba cv. Jinsixiaozao against Acetaminophen-Induced Liver Injury by Inhibiting Oxidative Stress and Inflammation in Mice

    doi: 10.3390/molecules22101781

    Figure Lengend Snippet: The effect of ZJF pretreatment on the nuclear factor kappa B p65 (NF-κB p65), nuclear factor erythroid 2-related factor 2 (Nrf-2), and NAD(P)H: quinone oxidoreductase 1 (NQO1 ) protein expression in response to the APAP-induced hepatotoxicity. ( A ) Western blotting analyses of NF-κB p65, Nrf-2, and NQO1 proteins; quantitative densitometric analyses of ( B ) NF-κB p65, ( C ) Nrf-2, and ( D ) NQO1 proteins normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each value represents the mean ± SEM of three independent experiments. ## p

    Article Snippet: APAP was produced by Aladdin chemistry Co. Ltd. (Shanghai, China) Anti-NF-κB p65 and Anti-NQO1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Western Blot

    Effects of ISL on the antioxidant system and ROS in HepG2 cells. A. After various time treatments with ISL, confocal microscopy was used to visualize the intracellular colocalization of Keap-1 (green) and Nrf-2 (red). B. After various time treatments with ISL, expression of NQO1 and HO-1 proteins were measured by western blot. β-Actin was used as a standard. C. Intracellular ROS levels were monitored by using DCFH-DA staining, and fluorescence was analyzed using flow cytometry. All data are expressed as mean ± SEM from three independent experiments. * p

    Journal: American Journal of Cancer Research

    Article Title: Disturbance of redox status enhances radiosensitivity of hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: Effects of ISL on the antioxidant system and ROS in HepG2 cells. A. After various time treatments with ISL, confocal microscopy was used to visualize the intracellular colocalization of Keap-1 (green) and Nrf-2 (red). B. After various time treatments with ISL, expression of NQO1 and HO-1 proteins were measured by western blot. β-Actin was used as a standard. C. Intracellular ROS levels were monitored by using DCFH-DA staining, and fluorescence was analyzed using flow cytometry. All data are expressed as mean ± SEM from three independent experiments. * p

    Article Snippet: The membrane was blocked for 1 h in TBST containing 0.5% FBS and subsequently probed with anti-Nrf2 antibody (Santa Cruz, CA, USA), anti-Keap1 antibody (Santa Cruz), anti-HO1 antibody (Cell Signaling, Danvers, USA) and anti-NQO1 antibody (Cell Signaling) at 4°C overnight with shaking.

    Techniques: Confocal Microscopy, Expressing, Western Blot, Staining, Fluorescence, Flow Cytometry, Cytometry

    The effect of DADS on the protein expression of nuclear factor kappa B p65 (NF-κB p65), i-κB, nuclear factor erythroid 2-related factor 2 (Nrf-2), and NAD(P)H: quinone oxidoreductase 1 (NQO1) ( A ) Western blotting analyses of NF-κB p65, i-κB, Nrf-2, and NQO1 proteins; quantitative densitometric analyses of ( B ) NF-κB p65; ( C ) i-κB; ( D ) Nrf-2; and ( E ) NQO1 proteins normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each value represents the mean ± SEM of three independent experiments. ### p

    Journal: Nutrients

    Article Title: Pharmacological Investigation of the Anti-Inflammation and Anti-Oxidation Activities of Diallyl Disulfide in a Rat Emphysema Model Induced by Cigarette Smoke Extract

    doi: 10.3390/nu10010079

    Figure Lengend Snippet: The effect of DADS on the protein expression of nuclear factor kappa B p65 (NF-κB p65), i-κB, nuclear factor erythroid 2-related factor 2 (Nrf-2), and NAD(P)H: quinone oxidoreductase 1 (NQO1) ( A ) Western blotting analyses of NF-κB p65, i-κB, Nrf-2, and NQO1 proteins; quantitative densitometric analyses of ( B ) NF-κB p65; ( C ) i-κB; ( D ) Nrf-2; and ( E ) NQO1 proteins normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each value represents the mean ± SEM of three independent experiments. ### p

    Article Snippet: Anti-NF-κB p65, i-κB, and NQO1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Western Blot

    Dicoumarol, an NQO1 inhibitor, blocks the apoptotic effects of β-lapachone. (A) CL1-1 cells (top) or CL1-5 cells (bottom) were left untreated or were incubated for 6 h with 5 µM β-lapachone and/or 10 µM dicoumarol, then stained with Annexin V-FITC and the Annexin V fluorescence measured by flow cytometry. (B) Morphological changes after drug treatment. CL1-1 or CL1-5 cells were left untreated (CTL) or were incubated for 24 h with 5 µM β-lapachone with or without 10 µM dicoumarol, then stained with acridine orange to observe the morphology of the cell nucleus. The scale bar represents 50 µm.

    Journal: PLoS ONE

    Article Title: Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    doi: 10.1371/journal.pone.0088122

    Figure Lengend Snippet: Dicoumarol, an NQO1 inhibitor, blocks the apoptotic effects of β-lapachone. (A) CL1-1 cells (top) or CL1-5 cells (bottom) were left untreated or were incubated for 6 h with 5 µM β-lapachone and/or 10 µM dicoumarol, then stained with Annexin V-FITC and the Annexin V fluorescence measured by flow cytometry. (B) Morphological changes after drug treatment. CL1-1 or CL1-5 cells were left untreated (CTL) or were incubated for 24 h with 5 µM β-lapachone with or without 10 µM dicoumarol, then stained with acridine orange to observe the morphology of the cell nucleus. The scale bar represents 50 µm.

    Article Snippet: The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA.

    Techniques: Incubation, Staining, Fluorescence, Flow Cytometry, Cytometry, CTL Assay

    β-lapachone-induced cell death is associated with NQO1 expression levels. (A) Percentage survival of the lung cancer cell lines CL1-1, CL1-5, and A549. Cells were treated with 0–10 µM β-lapachone for 12 h, then cell viability was determined by crystal violet staining assay and expressed as a percentage of the value for cultures with no β-lapachone. (B–D) NQO1 activity levels (B), NQO1 RNA expression levels (C), and NQO1 protein expression levels (D) in the three lung cancer cell lines grown under normal culture conditions. (E) Percentage survival of A549 cells (left panel), CL1-1 cells (center panel), and CL1-5 cells (right panel) incubated with the indicated concentration of β-lapachone for 3, 6, 12, or 24 h examined by crystal violet staining and expressed as percentage survival compared to the untreated cells. The results are the mean ± SD for 3 independent experiments, each in triplicate.

    Journal: PLoS ONE

    Article Title: Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    doi: 10.1371/journal.pone.0088122

    Figure Lengend Snippet: β-lapachone-induced cell death is associated with NQO1 expression levels. (A) Percentage survival of the lung cancer cell lines CL1-1, CL1-5, and A549. Cells were treated with 0–10 µM β-lapachone for 12 h, then cell viability was determined by crystal violet staining assay and expressed as a percentage of the value for cultures with no β-lapachone. (B–D) NQO1 activity levels (B), NQO1 RNA expression levels (C), and NQO1 protein expression levels (D) in the three lung cancer cell lines grown under normal culture conditions. (E) Percentage survival of A549 cells (left panel), CL1-1 cells (center panel), and CL1-5 cells (right panel) incubated with the indicated concentration of β-lapachone for 3, 6, 12, or 24 h examined by crystal violet staining and expressed as percentage survival compared to the untreated cells. The results are the mean ± SD for 3 independent experiments, each in triplicate.

    Article Snippet: The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA.

    Techniques: Expressing, Staining, Activity Assay, RNA Expression, Incubation, Concentration Assay

    NQO1 siRNA transfection significantly inhibits the effect of sulindac and its metabolites on β-lapachone-induced cell death. CL1-1 cells (left) or CL1-5 cells (right) were transfected with control siRNA (−) or NQO1 siRNA (+) for 24 h, then were left untreated or were incubated for 6 h with 100 or 250 µM sulindac (A), sulindac sulfone (B), or sulindac sulfide (C), then 2 µM β-lapachone or medium was added and the cells incubated for 12 h, when cell survival was measured using crystal violet staining and expressed as percentage survival compared to the untreated cells. * : p

    Journal: PLoS ONE

    Article Title: Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    doi: 10.1371/journal.pone.0088122

    Figure Lengend Snippet: NQO1 siRNA transfection significantly inhibits the effect of sulindac and its metabolites on β-lapachone-induced cell death. CL1-1 cells (left) or CL1-5 cells (right) were transfected with control siRNA (−) or NQO1 siRNA (+) for 24 h, then were left untreated or were incubated for 6 h with 100 or 250 µM sulindac (A), sulindac sulfone (B), or sulindac sulfide (C), then 2 µM β-lapachone or medium was added and the cells incubated for 12 h, when cell survival was measured using crystal violet staining and expressed as percentage survival compared to the untreated cells. * : p

    Article Snippet: The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA.

    Techniques: Transfection, Incubation, Staining

    The knockdown effects of NQO1 siRNA on NQO1 RNA, protein, and activity. (A–C) CL1-1 cells (left) or CL1-5 cells (right) were transfected for 1 to 3 days with control siRNA (CTL) or siRNA targeting NQO1, then RNA expression was measured by PCR (A) and protein expression by Western blotting (B and C). (D) CL1-1 cells transfected for 2 days with control siRNA or NQO1 siRNA were incubated alone or with 100 or 250 µM sulindac, sulindac sulfide, or sulindac sulfone for 6 or 24 h, then NQO1 activity was measured. * : p

    Journal: PLoS ONE

    Article Title: Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    doi: 10.1371/journal.pone.0088122

    Figure Lengend Snippet: The knockdown effects of NQO1 siRNA on NQO1 RNA, protein, and activity. (A–C) CL1-1 cells (left) or CL1-5 cells (right) were transfected for 1 to 3 days with control siRNA (CTL) or siRNA targeting NQO1, then RNA expression was measured by PCR (A) and protein expression by Western blotting (B and C). (D) CL1-1 cells transfected for 2 days with control siRNA or NQO1 siRNA were incubated alone or with 100 or 250 µM sulindac, sulindac sulfide, or sulindac sulfone for 6 or 24 h, then NQO1 activity was measured. * : p

    Article Snippet: The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA.

    Techniques: Activity Assay, Transfection, CTL Assay, RNA Expression, Polymerase Chain Reaction, Expressing, Western Blot, Incubation

    The increase in β-lapachone-induced cell death caused by sulindac and its metabolites is blocked by the NQO1 inhibitor, dicoumarol. CL1-1 cells (left) or CL1-5 cells (right) were left untreated or were pretreated for 6 h with 100 or 250 µM sulindac (A), sulindac sulfone (B), or sulindac sulfide (C) with or without 10 µM dicoumarol, then were incubated for a further 12 h with or without addition of 2 µM β-lapachone, then cell survival was measured by crystal violet staining and expressed as percentage survival compared to the untreated cells. * : p

    Journal: PLoS ONE

    Article Title: Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    doi: 10.1371/journal.pone.0088122

    Figure Lengend Snippet: The increase in β-lapachone-induced cell death caused by sulindac and its metabolites is blocked by the NQO1 inhibitor, dicoumarol. CL1-1 cells (left) or CL1-5 cells (right) were left untreated or were pretreated for 6 h with 100 or 250 µM sulindac (A), sulindac sulfone (B), or sulindac sulfide (C) with or without 10 µM dicoumarol, then were incubated for a further 12 h with or without addition of 2 µM β-lapachone, then cell survival was measured by crystal violet staining and expressed as percentage survival compared to the untreated cells. * : p

    Article Snippet: The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA.

    Techniques: Incubation, Staining

    Sulindac and its metabolites increase NQO1 expression and activity. (A) CL1-1 cells (left) or CL1-5 cells (right) were left untreated or were incubated with 100 or 250 µM sulindac, sulindac sulfone, or sulindac sulfide for 6, 12, or 24 h, then protein levels were measured by Western blotting. (B–D) CL1-1 cells (left) or CL1-5 cells (right) were left untreated (Ctl) or were incubated with the indicated concentration of sulindac (B), sulindac sulfone (C), or sulindac sulfide (D) for the indicated time, then NQO1 activity was measured. * : p

    Journal: PLoS ONE

    Article Title: Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    doi: 10.1371/journal.pone.0088122

    Figure Lengend Snippet: Sulindac and its metabolites increase NQO1 expression and activity. (A) CL1-1 cells (left) or CL1-5 cells (right) were left untreated or were incubated with 100 or 250 µM sulindac, sulindac sulfone, or sulindac sulfide for 6, 12, or 24 h, then protein levels were measured by Western blotting. (B–D) CL1-1 cells (left) or CL1-5 cells (right) were left untreated (Ctl) or were incubated with the indicated concentration of sulindac (B), sulindac sulfone (C), or sulindac sulfide (D) for the indicated time, then NQO1 activity was measured. * : p

    Article Snippet: The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T), then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling), PI3 kinase or p-PI3 kinase (Millipore), AKT or p-AKT (Epitomics), ERK, p-ERK, JNK, or p-JNK (Cell Signalling),GAPDH (Genetex) or β-actin (Abcam) diluted 1∶1000 in 1% BSA.

    Techniques: Expressing, Activity Assay, Incubation, Western Blot, CTL Assay, Concentration Assay

    Kaplan-Meier analysis of DFS and OS rates in 150 NSCLC patients in relation to NQO1 protein expression. Patients of NSCLC with NQO1 positive expression had lower DFS ( A , P

    Journal: BMC Cancer

    Article Title: NQO1 protein expression predicts poor prognosis of non-small cell lung cancers

    doi: 10.1186/s12885-015-1227-8

    Figure Lengend Snippet: Kaplan-Meier analysis of DFS and OS rates in 150 NSCLC patients in relation to NQO1 protein expression. Patients of NSCLC with NQO1 positive expression had lower DFS ( A , P

    Article Snippet: After washing with PBS, cells were incubated with antibody against NQO1 (1:200, Cell Signaling Technology, Boston, USA) for 2 hours at 37°C, and followed the incubation by Alexa Fluor®488 goat anti-rabbit IgG (H + C) (A11008, Invitrogen, USA) respectively, for 1 hour at RT.

    Techniques: Expressing

    Kaplan-Meier analysis of OS rates in patients with or without NQO1 expressed NSCLC in prognostic factors. OS was assessed in NSCLC patients with T1-2 ( A , P = 0.002), T3-4 ( B , P = 0.002), LN metastasis (−) ( C , P = 0.002), LN metastasis (+) ( D , P = 0.553), I-II stage ( E , P = 0.016), and III-IV stage ( F , P = 0.050) concomitant with either positive- or negative-expression of NQO1.

    Journal: BMC Cancer

    Article Title: NQO1 protein expression predicts poor prognosis of non-small cell lung cancers

    doi: 10.1186/s12885-015-1227-8

    Figure Lengend Snippet: Kaplan-Meier analysis of OS rates in patients with or without NQO1 expressed NSCLC in prognostic factors. OS was assessed in NSCLC patients with T1-2 ( A , P = 0.002), T3-4 ( B , P = 0.002), LN metastasis (−) ( C , P = 0.002), LN metastasis (+) ( D , P = 0.553), I-II stage ( E , P = 0.016), and III-IV stage ( F , P = 0.050) concomitant with either positive- or negative-expression of NQO1.

    Article Snippet: After washing with PBS, cells were incubated with antibody against NQO1 (1:200, Cell Signaling Technology, Boston, USA) for 2 hours at 37°C, and followed the incubation by Alexa Fluor®488 goat anti-rabbit IgG (H + C) (A11008, Invitrogen, USA) respectively, for 1 hour at RT.

    Techniques: Expressing

    IF staining for NQO1 protein in A549 human lung cancer cells. NQO1 protein located in the cytoplasm of A549 cells (Red for NQO1, Green for Actin, and Blue for DAPI).

    Journal: BMC Cancer

    Article Title: NQO1 protein expression predicts poor prognosis of non-small cell lung cancers

    doi: 10.1186/s12885-015-1227-8

    Figure Lengend Snippet: IF staining for NQO1 protein in A549 human lung cancer cells. NQO1 protein located in the cytoplasm of A549 cells (Red for NQO1, Green for Actin, and Blue for DAPI).

    Article Snippet: After washing with PBS, cells were incubated with antibody against NQO1 (1:200, Cell Signaling Technology, Boston, USA) for 2 hours at 37°C, and followed the incubation by Alexa Fluor®488 goat anti-rabbit IgG (H + C) (A11008, Invitrogen, USA) respectively, for 1 hour at RT.

    Techniques: Staining

    IHC staining for NQO1 protein expression in lung tissues. (A) NQO1 protein was negative in normal lung tissues. (B) NQO1 protein was showed diffuse and strong positive staining in cytopalsm of lung SCC cells with LN metastasis. (C) NQO1 was weakly positive in lung SCC without LN metastasis. (D) Diffuse and strong positive NQO1 protein signal in lung adenocarcinoma. (E F) NQO1 protein staining is negative or weakly positive in lung adenocarcinoma. (Original magnification, 200× in A-F ).

    Journal: BMC Cancer

    Article Title: NQO1 protein expression predicts poor prognosis of non-small cell lung cancers

    doi: 10.1186/s12885-015-1227-8

    Figure Lengend Snippet: IHC staining for NQO1 protein expression in lung tissues. (A) NQO1 protein was negative in normal lung tissues. (B) NQO1 protein was showed diffuse and strong positive staining in cytopalsm of lung SCC cells with LN metastasis. (C) NQO1 was weakly positive in lung SCC without LN metastasis. (D) Diffuse and strong positive NQO1 protein signal in lung adenocarcinoma. (E F) NQO1 protein staining is negative or weakly positive in lung adenocarcinoma. (Original magnification, 200× in A-F ).

    Article Snippet: After washing with PBS, cells were incubated with antibody against NQO1 (1:200, Cell Signaling Technology, Boston, USA) for 2 hours at 37°C, and followed the incubation by Alexa Fluor®488 goat anti-rabbit IgG (H + C) (A11008, Invitrogen, USA) respectively, for 1 hour at RT.

    Techniques: Immunohistochemistry, Staining, Expressing

    SIN activated Nrf2 pathway in rat lung tissues. ( A ) Protein level of Nrf2, HO-1, and NQO-1 in the lung tissues of rats from different groups was measured by Western blot assay; ( B–D ) relative Nrf2, HO-1, and NQO-1 mRNA level in the lung tissues of rats from different groups was measured by qRT-PCR. Con – Control group (rats exposed to normoxia); Con-S – rats with normoxia exposure + sinomenine administration 41 μg kg −1 ∙min −1 for 25 days; CIH – rats exposed to CIH alone; CIH-S – rats with CIH exposure + sinomenine administration at 41 μg kg −1 ∙min −1 for 25 days. Data are reported as means ±SE. ** p

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Sinomenine Attenuates Chronic Intermittent Hypoxia-Induced Lung Injury by Inhibiting Inflammation and Oxidative Stress

    doi: 10.12659/MSM.906577

    Figure Lengend Snippet: SIN activated Nrf2 pathway in rat lung tissues. ( A ) Protein level of Nrf2, HO-1, and NQO-1 in the lung tissues of rats from different groups was measured by Western blot assay; ( B–D ) relative Nrf2, HO-1, and NQO-1 mRNA level in the lung tissues of rats from different groups was measured by qRT-PCR. Con – Control group (rats exposed to normoxia); Con-S – rats with normoxia exposure + sinomenine administration 41 μg kg −1 ∙min −1 for 25 days; CIH – rats exposed to CIH alone; CIH-S – rats with CIH exposure + sinomenine administration at 41 μg kg −1 ∙min −1 for 25 days. Data are reported as means ±SE. ** p

    Article Snippet: Subsequently, the membranes were blocked with non-fat milk, incubated with primary antibodies (anti-β-actin, anti-p-p38, anti-NF-κB (p65), anti-Nox2, anti-Nrf2, anti-HO-1, and anti-NQO-1; with a 1: 1000 dilution for all (Cell Signaling Technology, Inc., Danvers, MA, USA), washed, and then incubated with a secondary antibody (1: 5000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA) at room temperature for 2 h. The protein blots were finally visualized using the chemiluminescent ECL reagent (Millipore, MA, USA).

    Techniques: Western Blot, Quantitative RT-PCR

    KOK up-regulates Keap1-Nrf2 signaling in the SNpc and striatum following MPTP intoxication. (A) At 7 days after the last MPTP intoxication, SNpc and striatal sections ( n = 5 per group) were subjected to MitoSOX staining. The staining intensity was then quantified. Insets display high magnification micrographs of areas marked with squares. (B,C) Seven days after the last MPTP intoxication, SNpc, and striatum sample ( n = 3 per group) were performed by Western blot assay. Keap1, Nrf2, HO-1, and NQO1. SNpc (B) and striatum (C) . The top panels illustrate representative Western blots. ANOVA test; ## p

    Journal: Frontiers in Pharmacology

    Article Title: Neuroprotective Effects of a Traditional Multi-Herbal Medicine Kyung-Ok-Ko in an Animal Model of Parkinson's Disease: Inhibition of MAPKs and NF-κB Pathways and Activation of Keap1-Nrf2 Pathway

    doi: 10.3389/fphar.2018.01444

    Figure Lengend Snippet: KOK up-regulates Keap1-Nrf2 signaling in the SNpc and striatum following MPTP intoxication. (A) At 7 days after the last MPTP intoxication, SNpc and striatal sections ( n = 5 per group) were subjected to MitoSOX staining. The staining intensity was then quantified. Insets display high magnification micrographs of areas marked with squares. (B,C) Seven days after the last MPTP intoxication, SNpc, and striatum sample ( n = 3 per group) were performed by Western blot assay. Keap1, Nrf2, HO-1, and NQO1. SNpc (B) and striatum (C) . The top panels illustrate representative Western blots. ANOVA test; ## p

    Article Snippet: For double immunofluorescent staining, sections were incubated overnight at 4°C with a mixture of mouse anti-PECAM-1 (1:500; Santa Cruz Biotechnology) and rabbit anti-GFAP (1:1,000; Millipore) antibodies or a mixture of rabbit anti-Nrf2 (1:500; Santa Cruz Biotechnology) and mouse anti-NQO1 (1:1,000; Cell Signaling Technology) antibodies.

    Techniques: Staining, Western Blot

    Nrf2 inhibitor neutralizes the protective effect of KOK following MPTP intoxication . (A–C) Nrf2 inhibitor (ML385; 30 mg/kg, i.p.) or saline was i.p. injected to mouse once daily from 30 min before KOK treatment in MPTP-intoxication model ( n = 4–6 per group). Pole test (A) , rotarod performance test (B) , and nest building behavior test (C) were accomplished at 5, 7, and 1 days, respectively, after the last MPTP intoxication. (D,E) Seven days after the last MPTP intoxication, nucleus, and cytosol isolated from SNpc (D) and striatum (E) from all groups ( n = 4 per group) were subjected to Western blot assay to quantify changes in Nrf2 nuclear translocation and NQO1 expression, respectively. (F) Seven days after the last MPTP intoxication, SNpc and striatal sections ( n = 3 per brain) were subjected to immunofluorescence staining using Nrf2 and NQO1 antibodies. Scale bar = 50 μm. ANOVA test; # p

    Journal: Frontiers in Pharmacology

    Article Title: Neuroprotective Effects of a Traditional Multi-Herbal Medicine Kyung-Ok-Ko in an Animal Model of Parkinson's Disease: Inhibition of MAPKs and NF-κB Pathways and Activation of Keap1-Nrf2 Pathway

    doi: 10.3389/fphar.2018.01444

    Figure Lengend Snippet: Nrf2 inhibitor neutralizes the protective effect of KOK following MPTP intoxication . (A–C) Nrf2 inhibitor (ML385; 30 mg/kg, i.p.) or saline was i.p. injected to mouse once daily from 30 min before KOK treatment in MPTP-intoxication model ( n = 4–6 per group). Pole test (A) , rotarod performance test (B) , and nest building behavior test (C) were accomplished at 5, 7, and 1 days, respectively, after the last MPTP intoxication. (D,E) Seven days after the last MPTP intoxication, nucleus, and cytosol isolated from SNpc (D) and striatum (E) from all groups ( n = 4 per group) were subjected to Western blot assay to quantify changes in Nrf2 nuclear translocation and NQO1 expression, respectively. (F) Seven days after the last MPTP intoxication, SNpc and striatal sections ( n = 3 per brain) were subjected to immunofluorescence staining using Nrf2 and NQO1 antibodies. Scale bar = 50 μm. ANOVA test; # p

    Article Snippet: For double immunofluorescent staining, sections were incubated overnight at 4°C with a mixture of mouse anti-PECAM-1 (1:500; Santa Cruz Biotechnology) and rabbit anti-GFAP (1:1,000; Millipore) antibodies or a mixture of rabbit anti-Nrf2 (1:500; Santa Cruz Biotechnology) and mouse anti-NQO1 (1:1,000; Cell Signaling Technology) antibodies.

    Techniques: Injection, Isolation, Western Blot, Translocation Assay, Expressing, Immunofluorescence, Staining

    Induction of HO-1 is not protective against ATO-induced apoptosis in MM cell lines. U266, MM.1s, 8226/S and KMS11 were treated for 0, 6, 24, and 48 h with 2 μ m ATO. Total protein lysates were obtained and NQO1 ( A ) and HO-1 ( B ) protein expression

    Journal: The Journal of Biological Chemistry

    Article Title: Reactive Oxygen Species Are Not Required for an Arsenic Trioxide-induced Antioxidant Response or Apoptosis *Reactive Oxygen Species Are Not Required for an Arsenic Trioxide-induced Antioxidant Response or Apoptosis * S⃞

    doi: 10.1074/jbc.M806546200

    Figure Lengend Snippet: Induction of HO-1 is not protective against ATO-induced apoptosis in MM cell lines. U266, MM.1s, 8226/S and KMS11 were treated for 0, 6, 24, and 48 h with 2 μ m ATO. Total protein lysates were obtained and NQO1 ( A ) and HO-1 ( B ) protein expression

    Article Snippet: Antibodies —The following primary antibodies were used: rabbit anti-HO-1 polyclonal antibody (pAb) (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-NQO1 monoclonal antibody (mAb) (Cell Signaling, Danvers, MA); rabbit anti-actin pAb (Sigma-Aldrich); rabbit anti-Nrf2 pAb (Santa Cruz Biotechnology); rabbit anti-Keap1 pAb (Proteintech Group Inc., Chicago, IL); rabbit anti-Lamin A/C pAb (Abcam, Cambridge, MA), and the mouse anti-Noxa mAb (Abcam).

    Techniques: Expressing

    NQO1 controls mitotic progression. (A) Control (ctr) siRNA and NQO1 siRNA transfected MCF-7 cells were synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h) followed by release in normal medium. Flow cytometric analysis of cell cycle at the indicated time points after release was done using propidium iodide DNA staining. (B) Table shows percentage of cells in each phase of the cell cycle (G0/G1, S, and G2/M) at indicated time points after nocodazole release from the experiment presented in (A). (C) Control shRNA and NQO1 shRNA stable MCF-7 cells were synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h). Cell cycle distribution was assessed as in A. (D) Mitotic progression of MCF-7 cells transfected with either control (ctr) siRNA or NQO1 siRNA was monitored by live-cell microscopy. Characteristic phase-contrast images are shown. Mitosis is completed in less than 90 min under normal conditions (arrow, upper). Mitosis fails to be completed as evidenced by the lack of formation of two daughter cells (arrow, lower). (E) MCF-7 cells stably expressing H2B-GFP were transfected with either control (ctr) siRNA or NQO1 siRNA. Mitosis was monitored by live-cell microscopy. Characteristic images are shown. Normal completion of mitosis is shown in the upper panel, whereas impaired mitoses are shown in the lower panel. Scale bar is 10 µm. (F) Percentage of cells exhibiting mitotic defects under different experimental conditions. Mitoses were checked in control (ctr) siRNA, NQO1 siRNA and NQO1 siRNA treated with Nicotinamide mononucleotide (ΝΜΝ, 0.5 mΜ) MCF-7 cells.

    Journal: Free radical biology & medicine

    Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

    doi: 10.1016/j.freeradbiomed.2018.08.009

    Figure Lengend Snippet: NQO1 controls mitotic progression. (A) Control (ctr) siRNA and NQO1 siRNA transfected MCF-7 cells were synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h) followed by release in normal medium. Flow cytometric analysis of cell cycle at the indicated time points after release was done using propidium iodide DNA staining. (B) Table shows percentage of cells in each phase of the cell cycle (G0/G1, S, and G2/M) at indicated time points after nocodazole release from the experiment presented in (A). (C) Control shRNA and NQO1 shRNA stable MCF-7 cells were synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h). Cell cycle distribution was assessed as in A. (D) Mitotic progression of MCF-7 cells transfected with either control (ctr) siRNA or NQO1 siRNA was monitored by live-cell microscopy. Characteristic phase-contrast images are shown. Mitosis is completed in less than 90 min under normal conditions (arrow, upper). Mitosis fails to be completed as evidenced by the lack of formation of two daughter cells (arrow, lower). (E) MCF-7 cells stably expressing H2B-GFP were transfected with either control (ctr) siRNA or NQO1 siRNA. Mitosis was monitored by live-cell microscopy. Characteristic images are shown. Normal completion of mitosis is shown in the upper panel, whereas impaired mitoses are shown in the lower panel. Scale bar is 10 µm. (F) Percentage of cells exhibiting mitotic defects under different experimental conditions. Mitoses were checked in control (ctr) siRNA, NQO1 siRNA and NQO1 siRNA treated with Nicotinamide mononucleotide (ΝΜΝ, 0.5 mΜ) MCF-7 cells.

    Article Snippet: Cells were incubated with appropriate primary antibodies against NQO1 (Cell signaling, dilution 1:200), and SIRT2 (Sigma, dilution 1:200) followed by incubation with Alexa Fluor 488 or 568 secondary antibodies (Life Technologies).

    Techniques: Transfection, Flow Cytometry, Staining, shRNA, Microscopy, Stable Transfection, Expressing

    NQO1 and SIRT2 colocalize to the mitotic spindle during mitosis. (A, B) Confocal microscopy images show the locations of endogenous SIRT2 (red) and NQO1 (green) during both metaphase (A) and telophase (B) in MCF-7 human breast cancer cells. (C, D) Confocal microscopy images show the locations of endogenous SIRT2 (red) and NQO1 (green) during both metaphase (C) and telophase (D) in BxPC-3 human pancreatic cancer cells. In all panels above, DNA was stained with DAPI (blue). On the right, graphs represent signal intensity scans along the lines drawn for each panel. Scale bar, 20 µm. (E, F, G) Higher magnification images (60x) under same experimental conditions as shown in A, B.

    Journal: Free radical biology & medicine

    Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

    doi: 10.1016/j.freeradbiomed.2018.08.009

    Figure Lengend Snippet: NQO1 and SIRT2 colocalize to the mitotic spindle during mitosis. (A, B) Confocal microscopy images show the locations of endogenous SIRT2 (red) and NQO1 (green) during both metaphase (A) and telophase (B) in MCF-7 human breast cancer cells. (C, D) Confocal microscopy images show the locations of endogenous SIRT2 (red) and NQO1 (green) during both metaphase (C) and telophase (D) in BxPC-3 human pancreatic cancer cells. In all panels above, DNA was stained with DAPI (blue). On the right, graphs represent signal intensity scans along the lines drawn for each panel. Scale bar, 20 µm. (E, F, G) Higher magnification images (60x) under same experimental conditions as shown in A, B.

    Article Snippet: Cells were incubated with appropriate primary antibodies against NQO1 (Cell signaling, dilution 1:200), and SIRT2 (Sigma, dilution 1:200) followed by incubation with Alexa Fluor 488 or 568 secondary antibodies (Life Technologies).

    Techniques: Confocal Microscopy, Staining

    NQO1 positively regulates SIRT2 activity. (A) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA. 48 h after transfection, lysates were analyzed by western blotting using antibodies against Lys-40 acetylated tubulin (Ac-Tubulin), NQO1, SIRT2 and tubulin/actin. Quantification of acetylated tubulin levels are presented, **p

    Journal: Free radical biology & medicine

    Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

    doi: 10.1016/j.freeradbiomed.2018.08.009

    Figure Lengend Snippet: NQO1 positively regulates SIRT2 activity. (A) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA. 48 h after transfection, lysates were analyzed by western blotting using antibodies against Lys-40 acetylated tubulin (Ac-Tubulin), NQO1, SIRT2 and tubulin/actin. Quantification of acetylated tubulin levels are presented, **p

    Article Snippet: Cells were incubated with appropriate primary antibodies against NQO1 (Cell signaling, dilution 1:200), and SIRT2 (Sigma, dilution 1:200) followed by incubation with Alexa Fluor 488 or 568 secondary antibodies (Life Technologies).

    Techniques: Activity Assay, Transfection, Western Blot

    NQO1-SIRT2 axis regulates APC/C complex during mitosis. (A) Cell extracts from control shRNA (sh ctr) and NQO1 shRNA (sh NQO1) MCF-7 cells were used to immunoprecipitate endogenous Cdc27. Interaction with several components of the APC/C complex was determined by western blotting using the indicated antibodies. Specificity was confirmed by using species-matched control IgG as a negative control. Input levels of the interacting proteins are shown (right). (B) MCF-7 cells overexpressing HA-Cdh1 were transfected with either control (ctr) siRNA or NQO1 siRNA followed by immunoprecipitation using an anti-acetylated lysine antibody (Ac-K). Acetylated levels of Cdh1 were checked by western blotting using an anti-HA antibody. Specificity was confirmed by using species-matched control IgG as a negative control. In parallel, immunoprecipitation with an antibody against Cdc27 was performed to check interaction between Cdc27 and Cdh1. Input levels of both Cdc27 and NQO1 are shown. (C) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA followed by immunoprecipitation using an anti-acetylated lysine antibody (Ac-K). Acetylated levels of endogenous Cdh1 were checked by western blotting using an anti-Cdh1 antibody. Specificity was confirmed by using species-matched control IgG as a negative control. In parallel, immunoprecipitation with an antibody against Cdc27 was performed to check interaction between Cdc27 and Cdh1. Input levels of Cdh1, Cdc27 and NQO1 are shown. (D) Western blot analysis in whole cell lysates MCF-7 cells transfected with either control (ctr) siRNA or NQO1 siRNA. Antibodies against well–established mitotic proteins including Aurora A, Cyclin B1 and Cdc20 were used. Mitotic cells following nocodazole treatment were collected by shake-off, replated and harvested at indicated time points. Protein levels of SIRT2, NQO1 and actin are shown. (E) Quantification of Aurora A protein levels in (D) using the ImageJ software is presented. (F) For in vivo ubiquitination assay, Myc-Aurora A and HA-Ubiquitin were co-transfected into control shRNA (sh ctr) and NQO1 shRNA (sh NQO1) MCF-7 cells. To block proteasomal degradation, cells were treated with MG132 (10 µM, 4 h). Aurora A was immunoprecipitated using an anti-Myc antibody followed by western blotting against HA to detect ubiquitinated levels of Aurora A. Levels of NQO1 and actin are shown. (G) A similar in vivo ubiquitination assay was performed in MCF-7 cells transfected with either a control (ctr) vector or a Flag- NQO1 vector. Aurora A was immunoprecipitated using an anti-Myc antibody followed by western blotting against HA to detect ubiquitinated levels of Aurora A. Levels of Flag-NQO1 and actin are shown.

    Journal: Free radical biology & medicine

    Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

    doi: 10.1016/j.freeradbiomed.2018.08.009

    Figure Lengend Snippet: NQO1-SIRT2 axis regulates APC/C complex during mitosis. (A) Cell extracts from control shRNA (sh ctr) and NQO1 shRNA (sh NQO1) MCF-7 cells were used to immunoprecipitate endogenous Cdc27. Interaction with several components of the APC/C complex was determined by western blotting using the indicated antibodies. Specificity was confirmed by using species-matched control IgG as a negative control. Input levels of the interacting proteins are shown (right). (B) MCF-7 cells overexpressing HA-Cdh1 were transfected with either control (ctr) siRNA or NQO1 siRNA followed by immunoprecipitation using an anti-acetylated lysine antibody (Ac-K). Acetylated levels of Cdh1 were checked by western blotting using an anti-HA antibody. Specificity was confirmed by using species-matched control IgG as a negative control. In parallel, immunoprecipitation with an antibody against Cdc27 was performed to check interaction between Cdc27 and Cdh1. Input levels of both Cdc27 and NQO1 are shown. (C) MCF-7 cells were transfected with either control (ctr) siRNA or NQO1 siRNA followed by immunoprecipitation using an anti-acetylated lysine antibody (Ac-K). Acetylated levels of endogenous Cdh1 were checked by western blotting using an anti-Cdh1 antibody. Specificity was confirmed by using species-matched control IgG as a negative control. In parallel, immunoprecipitation with an antibody against Cdc27 was performed to check interaction between Cdc27 and Cdh1. Input levels of Cdh1, Cdc27 and NQO1 are shown. (D) Western blot analysis in whole cell lysates MCF-7 cells transfected with either control (ctr) siRNA or NQO1 siRNA. Antibodies against well–established mitotic proteins including Aurora A, Cyclin B1 and Cdc20 were used. Mitotic cells following nocodazole treatment were collected by shake-off, replated and harvested at indicated time points. Protein levels of SIRT2, NQO1 and actin are shown. (E) Quantification of Aurora A protein levels in (D) using the ImageJ software is presented. (F) For in vivo ubiquitination assay, Myc-Aurora A and HA-Ubiquitin were co-transfected into control shRNA (sh ctr) and NQO1 shRNA (sh NQO1) MCF-7 cells. To block proteasomal degradation, cells were treated with MG132 (10 µM, 4 h). Aurora A was immunoprecipitated using an anti-Myc antibody followed by western blotting against HA to detect ubiquitinated levels of Aurora A. Levels of NQO1 and actin are shown. (G) A similar in vivo ubiquitination assay was performed in MCF-7 cells transfected with either a control (ctr) vector or a Flag- NQO1 vector. Aurora A was immunoprecipitated using an anti-Myc antibody followed by western blotting against HA to detect ubiquitinated levels of Aurora A. Levels of Flag-NQO1 and actin are shown.

    Article Snippet: Cells were incubated with appropriate primary antibodies against NQO1 (Cell signaling, dilution 1:200), and SIRT2 (Sigma, dilution 1:200) followed by incubation with Alexa Fluor 488 or 568 secondary antibodies (Life Technologies).

    Techniques: shRNA, Western Blot, Negative Control, Transfection, Immunoprecipitation, Software, In Vivo, Ubiquitin Assay, Blocking Assay, Plasmid Preparation

    NQO1 interacts directly with SIRT2. (A) MCF-7 cells were transiently transfected with Flag- NQO1 and HA- SIRT2 . 48 h after transfection, cell lysates were subjected to immunoprecipitation (IP) using either a Flag (left) or an HA (right) antibody followed by western blotting using antibodies against HA and Flag, respectively. Specificity was confirmed by using species-matched control IgG as a negative control. (B) Cell lysates from MCF-7 cells were subjected to immunoprecipitation (IP) using a SIRT2 antibody followed by western blotting using an anti-NQO1 antibody. * denotes Ig heavy and light chain. (C) Cell lysates from MCF-7 cells as described in (A) either unsynchronized or synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h) were subjected to immunoprecipitation using an antibody against HA followed by western blotting using a Flag antibody. Specificity was confirmed by using species-matched control IgG as a negative control. Overexpression of both Flag- NQO1 and HA- SIRT2 is confirmed by western blotting (lower). (D) Reciprocal co-immunoprecipitation of endogenous SIRT2 and NQO1 in either unsynchronized or synchronized in mitosis Hela cells. For cell synchronization, HeLa cells were serum starved for 48 h and then released into regular media for 16 h. Cell lysates were subjected to immunoprecipitation (IP) using either a SIRT2 (left) or an NQO1 (right) antibody followed by western blotting using antibodies against SIRT2 and NQO1. Specificity was confirmed by using species-matched control IgG as a negative control. (E) Either unsynchronized or synchronized in mitosis (syn, as described in C) MCF-7 cells were fractionated into cytoplasmic extracts (cyt) and nuclear extracts (nuc). Levels of both SIRT2 and NQO1 were detected by western blotting. Successful cellular fractionation was confirmed by western blotting using antibodies against markers such as Lamin A/C (nuc) and actin (cyt). (F) Recombinant human SIRT2 and GST-NQO1 were used to check protein-protein interaction in vitro . Coomassie staining shows the molecular weight of the two proteins. GST-NQO1 was immunoprecipitated followed by SIRT2 immunodetection of (upper). In addition, SIRT2 was immunoprecipitated first, followed by immunodetection of GST-NQO1 (lower). Beads alone were used in the pull-down assays as negative controls.

    Journal: Free radical biology & medicine

    Article Title: NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity

    doi: 10.1016/j.freeradbiomed.2018.08.009

    Figure Lengend Snippet: NQO1 interacts directly with SIRT2. (A) MCF-7 cells were transiently transfected with Flag- NQO1 and HA- SIRT2 . 48 h after transfection, cell lysates were subjected to immunoprecipitation (IP) using either a Flag (left) or an HA (right) antibody followed by western blotting using antibodies against HA and Flag, respectively. Specificity was confirmed by using species-matched control IgG as a negative control. (B) Cell lysates from MCF-7 cells were subjected to immunoprecipitation (IP) using a SIRT2 antibody followed by western blotting using an anti-NQO1 antibody. * denotes Ig heavy and light chain. (C) Cell lysates from MCF-7 cells as described in (A) either unsynchronized or synchronized in mitosis after treatment with nocodazole (Noc, 200 nM for 12 h) were subjected to immunoprecipitation using an antibody against HA followed by western blotting using a Flag antibody. Specificity was confirmed by using species-matched control IgG as a negative control. Overexpression of both Flag- NQO1 and HA- SIRT2 is confirmed by western blotting (lower). (D) Reciprocal co-immunoprecipitation of endogenous SIRT2 and NQO1 in either unsynchronized or synchronized in mitosis Hela cells. For cell synchronization, HeLa cells were serum starved for 48 h and then released into regular media for 16 h. Cell lysates were subjected to immunoprecipitation (IP) using either a SIRT2 (left) or an NQO1 (right) antibody followed by western blotting using antibodies against SIRT2 and NQO1. Specificity was confirmed by using species-matched control IgG as a negative control. (E) Either unsynchronized or synchronized in mitosis (syn, as described in C) MCF-7 cells were fractionated into cytoplasmic extracts (cyt) and nuclear extracts (nuc). Levels of both SIRT2 and NQO1 were detected by western blotting. Successful cellular fractionation was confirmed by western blotting using antibodies against markers such as Lamin A/C (nuc) and actin (cyt). (F) Recombinant human SIRT2 and GST-NQO1 were used to check protein-protein interaction in vitro . Coomassie staining shows the molecular weight of the two proteins. GST-NQO1 was immunoprecipitated followed by SIRT2 immunodetection of (upper). In addition, SIRT2 was immunoprecipitated first, followed by immunodetection of GST-NQO1 (lower). Beads alone were used in the pull-down assays as negative controls.

    Article Snippet: Cells were incubated with appropriate primary antibodies against NQO1 (Cell signaling, dilution 1:200), and SIRT2 (Sigma, dilution 1:200) followed by incubation with Alexa Fluor 488 or 568 secondary antibodies (Life Technologies).

    Techniques: Transfection, Immunoprecipitation, Western Blot, Negative Control, Over Expression, Cell Fractionation, Recombinant, In Vitro, Staining, Molecular Weight, Immunodetection

    The schematic diagram of the antioxidant molecular mechanisms of matrine‐type alkaloids. AGE s (advanced glycation end products) trigger intracellular ROS (reactive oxygen species) production, which induces apoptosis of aortic endothelial cells. The matrine‐type alkaloids administration promotes the phosphorylation of MKK 3 and MKK 6. As the kinases of p38 MAPK , phosphorylated MKK 3 and MKK 6 further phosphorylate p38 MAPK, which facilitate the nuclear translocation of Nrf2. Binding with ARE (antioxidant response element), the nuclear transcription factor Nrf2 initiates the transcriptions of antioxidant proteins such as NQO 1 (NADPH quinone oxidoreductase 1) and HO 1 (heme oxygenase 1). Thus, the intracellular ROS accumulation is alleviated and apoptosis is suppressed.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Matrine‐Type Alkaloids Inhibit Advanced Glycation End Products Induced Reactive Oxygen Species‐Mediated Apoptosis of Aortic Endothelial Cells In Vivo and In Vitro by Targeting MKK3 and p38MAPK Signaling

    doi: 10.1161/JAHA.117.007441

    Figure Lengend Snippet: The schematic diagram of the antioxidant molecular mechanisms of matrine‐type alkaloids. AGE s (advanced glycation end products) trigger intracellular ROS (reactive oxygen species) production, which induces apoptosis of aortic endothelial cells. The matrine‐type alkaloids administration promotes the phosphorylation of MKK 3 and MKK 6. As the kinases of p38 MAPK , phosphorylated MKK 3 and MKK 6 further phosphorylate p38 MAPK, which facilitate the nuclear translocation of Nrf2. Binding with ARE (antioxidant response element), the nuclear transcription factor Nrf2 initiates the transcriptions of antioxidant proteins such as NQO 1 (NADPH quinone oxidoreductase 1) and HO 1 (heme oxygenase 1). Thus, the intracellular ROS accumulation is alleviated and apoptosis is suppressed.

    Article Snippet: Specific antibodies against Nrf2 (1:500Cell Signaling Technology), MKK3 (1:500; Sigma‐Aldrich), phospho‐MKK3 (1:500; Sigma‐Aldrich), MKK6 (1:500; Sigma‐Aldrich), phospho‐MKK6 (1:500; Sigma‐Aldrich), p38 (1:250; Cell Signaling Technology), phospho‐p38 (1:250; Cell Signaling Technology), heme oxygenase (HO1; 1:1000; Cell Signaling Technology), NADPH quinone oxidoreductase (NQO1; 1:500; Cell Signaling Technology), histone H3 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), and GAPDH (1:1000; Invitrogen) were used to incubate membranes at 4°C for 10 hours.

    Techniques: Translocation Assay, Binding Assay

    A, The immunoblots of Nrf2 in nuclear protein when histone H3 was used as the internal reference. B, The immunoblots of phosphorylated MKK 3 (p‐ MKK 3), MKK 3, p‐ MKK 6, MKK 6, p‐p38, p38, HO 1 (heme oxygenase 1), and NQO 1 (NADPH quinone oxidoreductase 1) in total protein when GAPDH was introduced as the internal reference. C through H, Columns in these panels indicate the relative expression level of Nrf2 (Nrf2/histone H3), phosphorylation level of MKK 3 (p‐ MKK 3/ MKK 3), phosporylation level of MKK 6 (p‐ MKK 6/ MKK 6), phosphorylation level of p38 MAPK , the relative expression levels of HO 1 ( HO 1/ GAPDH ), and the relative expression level of NQO 1 ( NQO 1/ GAPDH ), respectively (3 independent experiments were performed; * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Matrine‐Type Alkaloids Inhibit Advanced Glycation End Products Induced Reactive Oxygen Species‐Mediated Apoptosis of Aortic Endothelial Cells In Vivo and In Vitro by Targeting MKK3 and p38MAPK Signaling

    doi: 10.1161/JAHA.117.007441

    Figure Lengend Snippet: A, The immunoblots of Nrf2 in nuclear protein when histone H3 was used as the internal reference. B, The immunoblots of phosphorylated MKK 3 (p‐ MKK 3), MKK 3, p‐ MKK 6, MKK 6, p‐p38, p38, HO 1 (heme oxygenase 1), and NQO 1 (NADPH quinone oxidoreductase 1) in total protein when GAPDH was introduced as the internal reference. C through H, Columns in these panels indicate the relative expression level of Nrf2 (Nrf2/histone H3), phosphorylation level of MKK 3 (p‐ MKK 3/ MKK 3), phosporylation level of MKK 6 (p‐ MKK 6/ MKK 6), phosphorylation level of p38 MAPK , the relative expression levels of HO 1 ( HO 1/ GAPDH ), and the relative expression level of NQO 1 ( NQO 1/ GAPDH ), respectively (3 independent experiments were performed; * P

    Article Snippet: Specific antibodies against Nrf2 (1:500Cell Signaling Technology), MKK3 (1:500; Sigma‐Aldrich), phospho‐MKK3 (1:500; Sigma‐Aldrich), MKK6 (1:500; Sigma‐Aldrich), phospho‐MKK6 (1:500; Sigma‐Aldrich), p38 (1:250; Cell Signaling Technology), phospho‐p38 (1:250; Cell Signaling Technology), heme oxygenase (HO1; 1:1000; Cell Signaling Technology), NADPH quinone oxidoreductase (NQO1; 1:500; Cell Signaling Technology), histone H3 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), and GAPDH (1:1000; Invitrogen) were used to incubate membranes at 4°C for 10 hours.

    Techniques: Western Blot, Expressing

    A, The immunoblots of Nrf2 in nuclear protein when histone H3 was used as the internal reference. B, The immunoblots of phosphorylated MKK 3 (p‐ MKK 3), MKK 3, p‐ MKK 6, MKK 6, p‐p38, p38, HO 1 (heme oxygenase 1), and NQO 1 (NADPH quinone oxidoreductase 1) in total protein when GAPDH was introduced as the internal reference. C through H, Columns in these panels indicate the relative expression level of Nrf2 (Nrf2/histon H3), phosphorylation level of MKK 3 (p‐ MKK 3/ MKK 3), phosporylation level of MKK 6 (p‐ MKK 6/ MKK 6), phosphorylation level of p38 MAPK , the relative expression levels of HO 1 ( HO 1/ GAPDH ), and the relative expression level of NQO 1 ( NQO 1/ GAPDH ), respectively (3 independent experiments were performed; * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Matrine‐Type Alkaloids Inhibit Advanced Glycation End Products Induced Reactive Oxygen Species‐Mediated Apoptosis of Aortic Endothelial Cells In Vivo and In Vitro by Targeting MKK3 and p38MAPK Signaling

    doi: 10.1161/JAHA.117.007441

    Figure Lengend Snippet: A, The immunoblots of Nrf2 in nuclear protein when histone H3 was used as the internal reference. B, The immunoblots of phosphorylated MKK 3 (p‐ MKK 3), MKK 3, p‐ MKK 6, MKK 6, p‐p38, p38, HO 1 (heme oxygenase 1), and NQO 1 (NADPH quinone oxidoreductase 1) in total protein when GAPDH was introduced as the internal reference. C through H, Columns in these panels indicate the relative expression level of Nrf2 (Nrf2/histon H3), phosphorylation level of MKK 3 (p‐ MKK 3/ MKK 3), phosporylation level of MKK 6 (p‐ MKK 6/ MKK 6), phosphorylation level of p38 MAPK , the relative expression levels of HO 1 ( HO 1/ GAPDH ), and the relative expression level of NQO 1 ( NQO 1/ GAPDH ), respectively (3 independent experiments were performed; * P

    Article Snippet: Specific antibodies against Nrf2 (1:500Cell Signaling Technology), MKK3 (1:500; Sigma‐Aldrich), phospho‐MKK3 (1:500; Sigma‐Aldrich), MKK6 (1:500; Sigma‐Aldrich), phospho‐MKK6 (1:500; Sigma‐Aldrich), p38 (1:250; Cell Signaling Technology), phospho‐p38 (1:250; Cell Signaling Technology), heme oxygenase (HO1; 1:1000; Cell Signaling Technology), NADPH quinone oxidoreductase (NQO1; 1:500; Cell Signaling Technology), histone H3 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), and GAPDH (1:1000; Invitrogen) were used to incubate membranes at 4°C for 10 hours.

    Techniques: Western Blot, Expressing