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  • 99
    Millipore keap1 polyclonal antibody
    Keap1 Polyclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc keap 1 p586 antibody
    Keap 1 P586 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Abcam anti keap 1 antibody
    Effects of the iodide intake (NI, 10 HI, and 100 HI) on the changes of (a) Nrf2; (b) Keap 1; (c) Srx; and (d) Prx 3 expressions on Days 7, 14, and 28. Representative western blot and histograms of densitometric analyses normalized for the relative  β -actin content. All data is presented as the mean ± SD ( N  = 6 for each group). Statistical analyses were performed by one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test.  ∗ p
    Anti Keap 1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology keap 1 sirna
    The effects of H 2 S on autophagy are attributed to <t>Keap-1.</t> H9C2 cells were treated with HG+Pal, HG+Pal+NaHS, HG+Pal+NaHS+PPG (10 nM, an irreversible competitive CSE inhibitor), HG+Pal+Mito-TEMPO (2 μ M, an inhibitor of mitochondrial ROS), HG+Pal+NAC (100 μ M, an inhibitor of ROS) and HG+Pal+NaHS+Keap-1 <t>siRNA</t> for 48 h. ( a ) The autophagosome was detected using an MDC test in H9C2 cells (green). Red arrows indicate autophagosome accumulation. ( b ) The expression of P62, ATG7, Beclin1 and LC3 II/I was detected by western blotting. ( c ) The effect of Keap-1 siRNA on autophagy was detected by western blotting. ( d ) The expression of Beclin1 and the ratio of LC3 II to LC3 I were detected by western blotting
    Keap 1 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher keap 1 sirna
    Nrf2 activation was correlated with M2 TEM formation and VEGF-α expression. a the <t>siRNA</t> of Nrf2 and <t>Keap-1</t> and DEM were used to pre-modulate Nrf2 activation. The nuclear Nrf2 were detected by western blotting. b the M1/2 macrophages markers of Nrf2 knocked-down (si-Nrf2) were detected after cancer cell CM stimulation ( n = 4). c 24-h secretion of IL-1, IL-6 and VEGF of Nrf2 knocked-down TEM were evaluated by ELISA ( n = 3). d the M1/2 macrophages markers of Keap-1 knocked-down (si-Keap-1) were detected after cancer cell CM stimulation ( n = 4). e IL-1, IL-6 and VEGF concentration of Keap-1 knocked-down (si-Keap-1) TEM were evaluated by ELISA ( n = 3). f M0 macrophages were pre-treated with 100-mM DEM for 6 h before cancer cell CM stimulation, the M1/2 macrophages markers were detected by PCR ( n = 4). g , IL-1, IL-6 and VEGF concentration of DEM pre-treated (DEM) were evaluated by ELISA ( n = 3). Graphs show the data as mean ± SD. *, P
    Keap 1 Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Proteintech anti keap 1
    Nrf2 activation was correlated with M2 TEM formation and VEGF-α expression. a the <t>siRNA</t> of Nrf2 and <t>Keap-1</t> and DEM were used to pre-modulate Nrf2 activation. The nuclear Nrf2 were detected by western blotting. b the M1/2 macrophages markers of Nrf2 knocked-down (si-Nrf2) were detected after cancer cell CM stimulation ( n = 4). c 24-h secretion of IL-1, IL-6 and VEGF of Nrf2 knocked-down TEM were evaluated by ELISA ( n = 3). d the M1/2 macrophages markers of Keap-1 knocked-down (si-Keap-1) were detected after cancer cell CM stimulation ( n = 4). e IL-1, IL-6 and VEGF concentration of Keap-1 knocked-down (si-Keap-1) TEM were evaluated by ELISA ( n = 3). f M0 macrophages were pre-treated with 100-mM DEM for 6 h before cancer cell CM stimulation, the M1/2 macrophages markers were detected by PCR ( n = 4). g , IL-1, IL-6 and VEGF concentration of DEM pre-treated (DEM) were evaluated by ELISA ( n = 3). Graphs show the data as mean ± SD. *, P
    Anti Keap 1, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Cell Signaling Technology Inc primary antibodies against keap 1
    Expression of <t>KEAP</t> 1 and Nrf2 in Nox4 tg mouse hearts. Immunocytochemistry showing stronger cytoplasmic expression of KEAP 1 in wt (A) compared to Nox4 tg (B) hearts (brown stain). Increased nuclear localisation of Nrf2 expression is seen in tg (D) compared to wt (C) hearts (brown stain). Arrows depict strong nuclear expression of Nrf2 that is not apparent in wt sections. Nucleii are stained with hematoxylin (violet) in all panels. Scale bar: 10 μm.
    Primary Antibodies Against Keap 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology mouse anti keap 1
    Effects of Tanshinone IIA on NOX4 and Nrf2/ARE signaling axis in lung tissues of silicosis rats. ( A ) Relative mRNA expression of NOX4, Nrf2, <t>Keap-1,</t> HO-1 and NQO-1 in lung tissues determined by RT-PCR analyses. ( B ) Representative bands of NOX4, Nrf2 in the cytoplasm and nucleus, Keap-1, HO-1 and NQO-1 as examined by western blotting; ( C ) Relative protein levels of NOX4, Nrf2, Keap-1, HO-1 and NQO-1 in lung tissues. Data are presented as the mean ± standard deviarion of three repeat experiments, n=6. *P
    Mouse Anti Keap 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc anti keap 1
    Gankyrin binds to the Kelch domain of <t>Keap1.</t> (A) Gankyrin influenced the binding of Keap1 to Nrf2. Equal amounts of cell lysates were immunoprecipitated with an anti-Keap1 antibody. Precipitated proteins and cell lysates were blotted with anti-Nrf2, anti-gankyrin, and anti-Keap1 antibodies. (B) Confocal microscopy was performed on HEK293T cells cotransfected with Keap1 and myc-gankyrin. Bar, 10 µm. (C and D) Gankyrin and Keap1 were cotransfected into 293T cells. Whole cell lysates were immunoprecipitated with Keap1- (C) or gankyrin-specific (D) antibodies. Precipitated proteins and cell lysates were blotted with the indicated antibodies. (E) Cell lysates from SMMC7721-con and SMMC7721-ovGank cells were immunoprecipitated with anti-Keap1 antibodies, and Western blot analysis was performed with the indicated antibodies. (F) The interaction of Myc-gankyrin with Flag-tagged truncated Keap1 fragments. The top panel shows a schematic of the truncated Keap1 fragments. HEK293T cells that were cotransfected with myc-gankyrin and Flag-tagged truncated Keap1 fragments were lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibodies. (G) The interaction of Flag-KC (Kelch domain of Keap1) with Myc-tagged gankyrin. HEK293T cells cotransfected with Flag-KC and Myc-tagged gankyrin were immunoprecipitated with anti-flag antibody and immunoblotted with anti-myc antibodies. (H) The interaction of Flag-Keap1 with Myc-tagged gankyrin mutants. The top panel shows a schematic of the gankyrin mutants. HEK293T cells cotransfected with Flag-Keap1 and myc-tagged deletion mutants of gankyrin were immunoprecipitated with anti-flag antibody. Precipitated proteins and cell lysates were blotted with anti-myc and the indicated antibodies. (I) Wild-type or ExxE motif-mutated gankyrin and Flag-Keap1 plasmids were transfected into HEK293T cells, and the cells were then lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibody. N-mutated indicated E in aa 21-24 were mutated to A, C-mutated indicated E in aa 201-204 were mutated to A, and N+C-mutated indicated E in aa 21-24 and aa 201-204 were all mutated. (J) The knockdown of Keap1 abolished the regulatory role of gankyrin on Nrf2 protein levels. Negative control oligonucleotides or siRNA targeting Keap1 were transfected into MHCCLM3-Con, -siGank, or SMMC7721-Con, -ovGank cells. Cell lysates were blotted with anti-Nrf2 and other indicated antibodies. (K) A coimmunoprecipitation assay was used to analyze the amount of gankyrin that was associated with Keap1 after stimulation with sulforaphane, tBHQ, or H 2 O 2 . SMMC7721 cells were stimulated by sulforaphane, tBHQ, or H 2 O 2 for 12 h, and the cells were then lysed and immunoprecipitated with an anti-Keap1 antibody. Precipitates and cell lysates were blotted with an anti-gankyrin antibody. The data are representative of at least two experiments with similar results.
    Anti Keap 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Xenobiotics keap 1 nrf2 interaction
    Links between <t>Nrf2</t> and FOXOs. Upper panel: reactive oxygen species (ROS), e.g. by interacting with <t>Keap-1,</t> trigger Nrf2 activation. As FOXO proteins control the expression of genes coding for antioxidant proteins, the activation of FOXO may, by blunting surges in levels of ROS, ameliorate the activity of Nrf2. Lower panel: FOXO3 was shown to also attenuate Nrf2 activity by transcriptionally upregulating the biosynthesis of Keap-1 [52] , which will bind and control Nrf2.
    Keap 1 Nrf2 Interaction, supplied by Xenobiotics, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Tocris keap 1 nrf 2 complex
    NADPH-derived reactive oxygen species is required for induction of heme oxygenase-1 (HO-1) in macrophages infected with Mycobacterium tuberculosis ( Mtb ) . (A) Human monocyte-derived macrophages were infected with Mtb H37Rv [multiplicity of infection (MOI): 3] in the presence or absence of an antioxidant (Tempol) for 24 h and HO-1 levels were measured in whole cell extracts by ELISA. Right panel shows HO-1 levels in infected macrophages treated with Tempol in the presence of a TAT-conjugated <t>NRF-2</t> sequence peptide that interacts with the <t>Keap-1/NRF-2</t> complex (TAT-14, 50 µM). (B) Generation of superoxide anions was quantified in cell supernatants of cultures shown in panel (A) . H 2 O 2 was used as positive control. (C) Activation of the HO-1 transcription factor NRF-2 in nuclear extracts was quantitatively assessed 12 h after infection using a colorimetric DNA-binding ELISA kit. (D) Bone marrow-differentiated macrophages were prepared from wild type (WT), p47phox −/− , or gp91phox −/− (deficient in two distinct subunits of the NADPH oxidase system) mice and infected in vitro with Mtb (MOI: 3) for 24 h. HO-1 levels were then assessed in whole cell extracts by ELISA. (E,F) HO-1 levels in cell extracts as well as activation of NRF-2 in nuclear extracts were quantified in macrophage cultures in the presence of TAT-14 (50 µM). Data are from at least three experiments using cells from a total of up to six healthy donors. (D–F) Three independent experiments were performed, with samples run in triplicates. Data from different biological groups were analyzed using the Kruskal–Wallis test, with the Dunn’s multiple-comparison test, whereas matched analyses were performed using the Wilcoxon matched-pairs test (* p
    Keap 1 Nrf 2 Complex, supplied by Tocris, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam anti keap 1
    Western blot analysis of Nrf-2 and <t>Keap-1</t> protein levels in the nucleus of sham, hsham, hovx, and hovxe groups of mice. Nrf-2 level is significantly increased in the nucleus of mice treated with hyperoxia alone. Small letters denote p values. ( a p =0.008, sham vs. hsham mice), as well as in mice treated with the combination of hyperoxia and ovariectomy ( b p =0.010, sham vs. hovx) in comparison to normoxic group of animals (A). Nuclear accumulation of Keap-1 remained unchanged regardless of any treatment (B). Representative immunoblots of Nrf-2 and Keap-1 proteins in nuclear fractions of sham, hsham, hovx and hovxe group of mice are shown. Histone H3 was used to assess the purity of nuclear fraction. Amidoblack was used as a loading control. The results are presented as mean±S.D. and normalized to sham group (C). The number of samples was n =2 per each group.
    Anti Keap 1, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc keap1
    EGb761 modulates oxidative stress response and oncogenic pathways. (A) Western blots for AKT, mTOR, ERK1/2, Nrf2 and <t>KEAP1</t> in 24h, 48h and 72h treated (+) versus untreated (-) cells are shown in representative images of three independent experiments. (B) Relative expression to control (untreated cells) is demonstrated as means ± SD by quantitative analysis using densitometry normalized to the corresponding beta-actin expression; *p
    Keap1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    WuXi AppTec keap1 antibody production
    <t>Keap1</t> ablation leads to ubiquitin aggregates accumulation and cytotoxicity. (A) Ubiquitin aggregates in Keap1 knockout cells. Keap1 +/+ and Keap1 −/− MEF cells were treated with 10 µg/ml puromycin for 4 hours, and recovered in normal
    Keap1 Antibody Production, supplied by WuXi AppTec, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    keap1  (Abcam)
    93
    Abcam keap1
    <t>Keap1</t> levels are unaltered while the concentration of Cullin-3 is decreased in EAE. (a) Keap1 protein levels in the spinal cord of control and EAE mice were determined by western blot analysis as described under “Materials and Methods”, and were normalized by the amount of coomassie blue stain in the same gel lane. Values represent the mean ± SEM of 3 animals per experimental group. Inset panel shows a representative western blot. (b) Cullin-3 levels in the spinal cord of control and EAE mice at 21 dpi were determined by western blot analysis as described under “Materials and Methods” and are expressed relative to those of GAPDH. Values represent the mean ± SEM of 3 animals per group. Asterisks denote values that are statistically different ( p
    Keap1, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Proteintech keap1
    The restorative effect of mitoQ on PINK and Parkin expression was mediated in part by <t>Nrf2/Keap1.</t> A: Immunoblotting assay for Keap1 and Nrf2 expression, which showed that mitoQ inhibited Keap1 and cytoplasmic Nrf2 expression in HK-2 cells exposed to HG and restored nuclear Nrf2 expression. A1-A3: Quantification of average Western blot band intensity. B: Immunofluorescence assay for Nrf2 showing decreased antibody staining intensity in HK-2 cells after incubation under HG conditions. MitoQ up-regulated the inhibition of Nrf2 intensity in HG-exposed cells. C: Bar graphs depicting Nrf2 binding to the ARE. D: The interaction between Keap1 and Nrf2 in HK-2 cells subjected to HG exposure with or without mitoQ was assessed using IP. D1 and D2: Quantification of the intensity of the interaction between Keap1 and Nrf2 using IP. E: Western blot analysis revealed that mitoQ restored PINK and Parkin expression in HK-2 cells exposed to HG, an effect that was partially abolished by transfection with Keap1 siRNA or Nrf2 siRNA. E1: Quantification of average Western blot band intensity. F: Similar results were observed for PINK mRNA expression, as demonstrated by real-time PCR. *P
    Keap1, supplied by Proteintech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological keap1 structure human keap1
    The restorative effect of mitoQ on PINK and Parkin expression was mediated in part by <t>Nrf2/Keap1.</t> A: Immunoblotting assay for Keap1 and Nrf2 expression, which showed that mitoQ inhibited Keap1 and cytoplasmic Nrf2 expression in HK-2 cells exposed to HG and restored nuclear Nrf2 expression. A1-A3: Quantification of average Western blot band intensity. B: Immunofluorescence assay for Nrf2 showing decreased antibody staining intensity in HK-2 cells after incubation under HG conditions. MitoQ up-regulated the inhibition of Nrf2 intensity in HG-exposed cells. C: Bar graphs depicting Nrf2 binding to the ARE. D: The interaction between Keap1 and Nrf2 in HK-2 cells subjected to HG exposure with or without mitoQ was assessed using IP. D1 and D2: Quantification of the intensity of the interaction between Keap1 and Nrf2 using IP. E: Western blot analysis revealed that mitoQ restored PINK and Parkin expression in HK-2 cells exposed to HG, an effect that was partially abolished by transfection with Keap1 siRNA or Nrf2 siRNA. E1: Quantification of average Western blot band intensity. F: Similar results were observed for PINK mRNA expression, as demonstrated by real-time PCR. *P
    Keap1 Structure Human Keap1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of the iodide intake (NI, 10 HI, and 100 HI) on the changes of (a) Nrf2; (b) Keap 1; (c) Srx; and (d) Prx 3 expressions on Days 7, 14, and 28. Representative western blot and histograms of densitometric analyses normalized for the relative  β -actin content. All data is presented as the mean ± SD ( N  = 6 for each group). Statistical analyses were performed by one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test.  ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Activation of the Nrf2-Keap 1 Pathway in Short-Term Iodide Excess in Thyroid in Rats

    doi: 10.1155/2017/4383652

    Figure Lengend Snippet: Effects of the iodide intake (NI, 10 HI, and 100 HI) on the changes of (a) Nrf2; (b) Keap 1; (c) Srx; and (d) Prx 3 expressions on Days 7, 14, and 28. Representative western blot and histograms of densitometric analyses normalized for the relative β -actin content. All data is presented as the mean ± SD ( N = 6 for each group). Statistical analyses were performed by one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test. ∗ p

    Article Snippet: Reagent Anti-Peroxiredoxin-3 antibody (ab16751) and anti-Keap 1 antibody (ab66620) were purchased from Abcam (Abcam, Cambridge, MA, USA).

    Techniques: Western Blot

    Proposed mechanisms in the present study. (a) The urinary iodine concentration and thyroid function of Wistar rats were detected. (b) The activation of the Nrf2-Keap 1 pathway induced by iodide excess in the thyroid. (c) The balance between oxidative stress and antioxidative defense under physiological conditions and excessive iodide intake.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Activation of the Nrf2-Keap 1 Pathway in Short-Term Iodide Excess in Thyroid in Rats

    doi: 10.1155/2017/4383652

    Figure Lengend Snippet: Proposed mechanisms in the present study. (a) The urinary iodine concentration and thyroid function of Wistar rats were detected. (b) The activation of the Nrf2-Keap 1 pathway induced by iodide excess in the thyroid. (c) The balance between oxidative stress and antioxidative defense under physiological conditions and excessive iodide intake.

    Article Snippet: Reagent Anti-Peroxiredoxin-3 antibody (ab16751) and anti-Keap 1 antibody (ab66620) were purchased from Abcam (Abcam, Cambridge, MA, USA).

    Techniques: Concentration Assay, Activation Assay

    MSU up-regulated the nuclear translocation of Nrf2 and caused lysosomal destabilization. THP-1 cells were incubated with or without 100 nM PMA for 3 h and were then treated with 75 µg/ml MSU for 6 h. ( a ) The levels of Nrf2 and Keap-1 in the nuclear

    Journal: Cellular and Molecular Immunology

    Article Title: Monosodium urate crystals trigger Nrf2- and heme oxygenase-1-dependent inflammation in THP-1 cells

    doi: 10.1038/cmi.2014.65

    Figure Lengend Snippet: MSU up-regulated the nuclear translocation of Nrf2 and caused lysosomal destabilization. THP-1 cells were incubated with or without 100 nM PMA for 3 h and were then treated with 75 µg/ml MSU for 6 h. ( a ) The levels of Nrf2 and Keap-1 in the nuclear

    Article Snippet: Keap-1 antibody was obtained from Abcam (Cambridge, MA, USA). p62 antibody was purchased from Novus (Littleton, CO, USA).

    Techniques: Translocation Assay, Incubation

    Confirmation of efficient KEAP1 knockdown in Hep2 cells. (A) mRNA expression levels in the three KEAP1 shRNA-transfected Hep2 cell groups. The highest reduction in KEAP1 mRNA levels compared with the scHep2 group was observed in the shKEAP1 group, with a decrease of 67±1%. (B) Western blot analysis demonstrated that the protein expression of KEAP1 was markedly reduced in the shKEAP1 group compared with parent Hep2 and scHep2 groups. (C) Green fluorescent protein fluorescence was observed to determine the transfection efficiency in shKEAP1 and scHep2 cells (magnification, ×40). (D) Immunofluorescence staining for KEAP1 demonstrated that KEAP1 protein expression was reduced in shKEAP1 cells compared with scHep2 and parental Hep2 cells. *P

    Journal: Molecular Medicine Reports

    Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells

    doi: 10.3892/mmr.2018.9288

    Figure Lengend Snippet: Confirmation of efficient KEAP1 knockdown in Hep2 cells. (A) mRNA expression levels in the three KEAP1 shRNA-transfected Hep2 cell groups. The highest reduction in KEAP1 mRNA levels compared with the scHep2 group was observed in the shKEAP1 group, with a decrease of 67±1%. (B) Western blot analysis demonstrated that the protein expression of KEAP1 was markedly reduced in the shKEAP1 group compared with parent Hep2 and scHep2 groups. (C) Green fluorescent protein fluorescence was observed to determine the transfection efficiency in shKEAP1 and scHep2 cells (magnification, ×40). (D) Immunofluorescence staining for KEAP1 demonstrated that KEAP1 protein expression was reduced in shKEAP1 cells compared with scHep2 and parental Hep2 cells. *P

    Article Snippet: To block the membranes, 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.4–7.5) was applied at room temperature for 1 h. Membranes were subsequently incubated overnight with anti-NRF2 (cat. no. ab31163; Abcam), anti-KEAP1 (cat. no. ab139729; Abcam), anti-NQO1 (cat. no. A180; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-HO1 (cat. no. ab68477; Abcam), and anti-β-actin (cat. no. GB13001-3; Servicebio, Inc.) or lamin B primary antibodies (GB11408; Servicebio, Inc.) in blocking buffer at 4°C at 1:1,000 dilution.

    Techniques: Expressing, shRNA, Transfection, Western Blot, Fluorescence, Immunofluorescence, Staining

    Effect of KEAP1 knockdown on the expression of NRF2 and downstream targets. (A) mRNA expression levels of NRF2, NQO1 and HO1 in Hep2, scHep2 and shKEAP1 Hep2 cells. Expression levels of NRF2, NQO1 and HO1 were increased following the knockdown of KEAP1 in Hep2 cells. (B) Representative NRF2 immunofluorescence staining images indicate that NRF2 translocated into the nuclei from the cytoplasm following knockdown of KEAP1 in Hep2 cells (magnification, ×40). (C) Western blotting demonstrated that nuclear NRF2 protein expression levels were elevated, while cytoplasmic NRF2 protein expression levels were reduced, following the knockdown of KEAP1 in Hep2 cells. (D) Western blotting demonstrated that total NQO1 and HO1 protein expression levels were increased within shKEAP1 Hep2 cells, compared with the scHep2 group. *P

    Journal: Molecular Medicine Reports

    Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells

    doi: 10.3892/mmr.2018.9288

    Figure Lengend Snippet: Effect of KEAP1 knockdown on the expression of NRF2 and downstream targets. (A) mRNA expression levels of NRF2, NQO1 and HO1 in Hep2, scHep2 and shKEAP1 Hep2 cells. Expression levels of NRF2, NQO1 and HO1 were increased following the knockdown of KEAP1 in Hep2 cells. (B) Representative NRF2 immunofluorescence staining images indicate that NRF2 translocated into the nuclei from the cytoplasm following knockdown of KEAP1 in Hep2 cells (magnification, ×40). (C) Western blotting demonstrated that nuclear NRF2 protein expression levels were elevated, while cytoplasmic NRF2 protein expression levels were reduced, following the knockdown of KEAP1 in Hep2 cells. (D) Western blotting demonstrated that total NQO1 and HO1 protein expression levels were increased within shKEAP1 Hep2 cells, compared with the scHep2 group. *P

    Article Snippet: To block the membranes, 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.4–7.5) was applied at room temperature for 1 h. Membranes were subsequently incubated overnight with anti-NRF2 (cat. no. ab31163; Abcam), anti-KEAP1 (cat. no. ab139729; Abcam), anti-NQO1 (cat. no. A180; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-HO1 (cat. no. ab68477; Abcam), and anti-β-actin (cat. no. GB13001-3; Servicebio, Inc.) or lamin B primary antibodies (GB11408; Servicebio, Inc.) in blocking buffer at 4°C at 1:1,000 dilution.

    Techniques: Expressing, Immunofluorescence, Staining, Western Blot

    Effect of KEAP1 knockdown on H 2 O 2 -induced alterations in the expression of NQO1 and HO1. mRNA levels of (A) NQO1 and (B) HO1 within shKEAP1 Hep2 cells were increased in a dose- and time-dependent manner following exposure to H 2 O 2 . KEAP1 knockdown upregulated the expression levels of NQO1 and HO1 compared with in scHep2 cells. *P

    Journal: Molecular Medicine Reports

    Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells

    doi: 10.3892/mmr.2018.9288

    Figure Lengend Snippet: Effect of KEAP1 knockdown on H 2 O 2 -induced alterations in the expression of NQO1 and HO1. mRNA levels of (A) NQO1 and (B) HO1 within shKEAP1 Hep2 cells were increased in a dose- and time-dependent manner following exposure to H 2 O 2 . KEAP1 knockdown upregulated the expression levels of NQO1 and HO1 compared with in scHep2 cells. *P

    Article Snippet: To block the membranes, 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.4–7.5) was applied at room temperature for 1 h. Membranes were subsequently incubated overnight with anti-NRF2 (cat. no. ab31163; Abcam), anti-KEAP1 (cat. no. ab139729; Abcam), anti-NQO1 (cat. no. A180; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-HO1 (cat. no. ab68477; Abcam), and anti-β-actin (cat. no. GB13001-3; Servicebio, Inc.) or lamin B primary antibodies (GB11408; Servicebio, Inc.) in blocking buffer at 4°C at 1:1,000 dilution.

    Techniques: Expressing

    Effect of KEAP1 knockdown on H 2 O 2 -induced alterations in Hep2 cell apoptosis. (A) H 2 O 2 evidently induced cell apoptosis from 14.1 to 27.9 and 31.8 to 45.3% in shKEAP1 and scHep2 cells, respectively. (B) The apoptotic rate in shKEAP1 Hep2 cells was lower compared with in scHep2 cells. *P

    Journal: Molecular Medicine Reports

    Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells

    doi: 10.3892/mmr.2018.9288

    Figure Lengend Snippet: Effect of KEAP1 knockdown on H 2 O 2 -induced alterations in Hep2 cell apoptosis. (A) H 2 O 2 evidently induced cell apoptosis from 14.1 to 27.9 and 31.8 to 45.3% in shKEAP1 and scHep2 cells, respectively. (B) The apoptotic rate in shKEAP1 Hep2 cells was lower compared with in scHep2 cells. *P

    Article Snippet: To block the membranes, 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.4–7.5) was applied at room temperature for 1 h. Membranes were subsequently incubated overnight with anti-NRF2 (cat. no. ab31163; Abcam), anti-KEAP1 (cat. no. ab139729; Abcam), anti-NQO1 (cat. no. A180; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-HO1 (cat. no. ab68477; Abcam), and anti-β-actin (cat. no. GB13001-3; Servicebio, Inc.) or lamin B primary antibodies (GB11408; Servicebio, Inc.) in blocking buffer at 4°C at 1:1,000 dilution.

    Techniques:

    The effects of H 2 S on autophagy are attributed to Keap-1. H9C2 cells were treated with HG+Pal, HG+Pal+NaHS, HG+Pal+NaHS+PPG (10 nM, an irreversible competitive CSE inhibitor), HG+Pal+Mito-TEMPO (2 μ M, an inhibitor of mitochondrial ROS), HG+Pal+NAC (100 μ M, an inhibitor of ROS) and HG+Pal+NaHS+Keap-1 siRNA for 48 h. ( a ) The autophagosome was detected using an MDC test in H9C2 cells (green). Red arrows indicate autophagosome accumulation. ( b ) The expression of P62, ATG7, Beclin1 and LC3 II/I was detected by western blotting. ( c ) The effect of Keap-1 siRNA on autophagy was detected by western blotting. ( d ) The expression of Beclin1 and the ratio of LC3 II to LC3 I were detected by western blotting

    Journal: Cell Death & Disease

    Article Title: Exogenous H2S facilitating ubiquitin aggregates clearance via autophagy attenuates type 2 diabetes-induced cardiomyopathy

    doi: 10.1038/cddis.2017.380

    Figure Lengend Snippet: The effects of H 2 S on autophagy are attributed to Keap-1. H9C2 cells were treated with HG+Pal, HG+Pal+NaHS, HG+Pal+NaHS+PPG (10 nM, an irreversible competitive CSE inhibitor), HG+Pal+Mito-TEMPO (2 μ M, an inhibitor of mitochondrial ROS), HG+Pal+NAC (100 μ M, an inhibitor of ROS) and HG+Pal+NaHS+Keap-1 siRNA for 48 h. ( a ) The autophagosome was detected using an MDC test in H9C2 cells (green). Red arrows indicate autophagosome accumulation. ( b ) The expression of P62, ATG7, Beclin1 and LC3 II/I was detected by western blotting. ( c ) The effect of Keap-1 siRNA on autophagy was detected by western blotting. ( d ) The expression of Beclin1 and the ratio of LC3 II to LC3 I were detected by western blotting

    Article Snippet: Keap-1 siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Western Blot

    Nrf2 activation was correlated with M2 TEM formation and VEGF-α expression. a the siRNA of Nrf2 and Keap-1 and DEM were used to pre-modulate Nrf2 activation. The nuclear Nrf2 were detected by western blotting. b the M1/2 macrophages markers of Nrf2 knocked-down (si-Nrf2) were detected after cancer cell CM stimulation ( n = 4). c 24-h secretion of IL-1, IL-6 and VEGF of Nrf2 knocked-down TEM were evaluated by ELISA ( n = 3). d the M1/2 macrophages markers of Keap-1 knocked-down (si-Keap-1) were detected after cancer cell CM stimulation ( n = 4). e IL-1, IL-6 and VEGF concentration of Keap-1 knocked-down (si-Keap-1) TEM were evaluated by ELISA ( n = 3). f M0 macrophages were pre-treated with 100-mM DEM for 6 h before cancer cell CM stimulation, the M1/2 macrophages markers were detected by PCR ( n = 4). g , IL-1, IL-6 and VEGF concentration of DEM pre-treated (DEM) were evaluated by ELISA ( n = 3). Graphs show the data as mean ± SD. *, P

    Journal: Cell Communication and Signaling : CCS

    Article Title: Nrf2 activation drive macrophages polarization and cancer cell epithelial-mesenchymal transition during interaction

    doi: 10.1186/s12964-018-0262-x

    Figure Lengend Snippet: Nrf2 activation was correlated with M2 TEM formation and VEGF-α expression. a the siRNA of Nrf2 and Keap-1 and DEM were used to pre-modulate Nrf2 activation. The nuclear Nrf2 were detected by western blotting. b the M1/2 macrophages markers of Nrf2 knocked-down (si-Nrf2) were detected after cancer cell CM stimulation ( n = 4). c 24-h secretion of IL-1, IL-6 and VEGF of Nrf2 knocked-down TEM were evaluated by ELISA ( n = 3). d the M1/2 macrophages markers of Keap-1 knocked-down (si-Keap-1) were detected after cancer cell CM stimulation ( n = 4). e IL-1, IL-6 and VEGF concentration of Keap-1 knocked-down (si-Keap-1) TEM were evaluated by ELISA ( n = 3). f M0 macrophages were pre-treated with 100-mM DEM for 6 h before cancer cell CM stimulation, the M1/2 macrophages markers were detected by PCR ( n = 4). g , IL-1, IL-6 and VEGF concentration of DEM pre-treated (DEM) were evaluated by ELISA ( n = 3). Graphs show the data as mean ± SD. *, P

    Article Snippet: Cells were transfected with Nrf2 siRNA (Applied Biosystems, s9492, Waltham, Massachusetts, USA), Keap-1 siRNA (Applied Biosystems, s18981, Waltham, MA, USA) or negative control siRNA (Applied Biosystems, Select Negative Control #1 siRNA, Waltham, MA, USA) at 10 nmol/L using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instructions.

    Techniques: Activation Assay, Transmission Electron Microscopy, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Polymerase Chain Reaction

    Expression of KEAP 1 and Nrf2 in Nox4 tg mouse hearts. Immunocytochemistry showing stronger cytoplasmic expression of KEAP 1 in wt (A) compared to Nox4 tg (B) hearts (brown stain). Increased nuclear localisation of Nrf2 expression is seen in tg (D) compared to wt (C) hearts (brown stain). Arrows depict strong nuclear expression of Nrf2 that is not apparent in wt sections. Nucleii are stained with hematoxylin (violet) in all panels. Scale bar: 10 μm.

    Journal: Free Radical Biology & Medicine

    Article Title: Nox4 regulates Nrf2 and glutathione redox in cardiomyocytes in vivo

    doi: 10.1016/j.freeradbiomed.2011.04.022

    Figure Lengend Snippet: Expression of KEAP 1 and Nrf2 in Nox4 tg mouse hearts. Immunocytochemistry showing stronger cytoplasmic expression of KEAP 1 in wt (A) compared to Nox4 tg (B) hearts (brown stain). Increased nuclear localisation of Nrf2 expression is seen in tg (D) compared to wt (C) hearts (brown stain). Arrows depict strong nuclear expression of Nrf2 that is not apparent in wt sections. Nucleii are stained with hematoxylin (violet) in all panels. Scale bar: 10 μm.

    Article Snippet: Primary antibodies against KEAP 1 (Cell Signaling Technology, Cat. No. 4617) and Nrf2 (Santa Cruz Biotechnology, Cat. No. sc-722) were both used at a dilution of 1:50.

    Techniques: Expressing, Immunocytochemistry, Staining

    Effects of Tanshinone IIA on NOX4 and Nrf2/ARE signaling axis in lung tissues of silicosis rats. ( A ) Relative mRNA expression of NOX4, Nrf2, Keap-1, HO-1 and NQO-1 in lung tissues determined by RT-PCR analyses. ( B ) Representative bands of NOX4, Nrf2 in the cytoplasm and nucleus, Keap-1, HO-1 and NQO-1 as examined by western blotting; ( C ) Relative protein levels of NOX4, Nrf2, Keap-1, HO-1 and NQO-1 in lung tissues. Data are presented as the mean ± standard deviarion of three repeat experiments, n=6. *P

    Journal: Drug Design, Development and Therapy

    Article Title: The Protective Role of Tanshinone IIA in Silicosis Rat Model via TGF-β1/Smad Signaling Suppression, NOX4 Inhibition and Nrf2/ARE Signaling Activation

    doi: 10.2147/DDDT.S230572

    Figure Lengend Snippet: Effects of Tanshinone IIA on NOX4 and Nrf2/ARE signaling axis in lung tissues of silicosis rats. ( A ) Relative mRNA expression of NOX4, Nrf2, Keap-1, HO-1 and NQO-1 in lung tissues determined by RT-PCR analyses. ( B ) Representative bands of NOX4, Nrf2 in the cytoplasm and nucleus, Keap-1, HO-1 and NQO-1 as examined by western blotting; ( C ) Relative protein levels of NOX4, Nrf2, Keap-1, HO-1 and NQO-1 in lung tissues. Data are presented as the mean ± standard deviarion of three repeat experiments, n=6. *P

    Article Snippet: After blocking with 5% non-fat milk made with Tris-buffered saline (containing 0.1% Tween 20), the membranes were incubated with the following specific primary antibodies at 4°C overnight: rabbit anti-TGF-β1 (1:1000, abcam; cat. no. ab-92486), rabbit anti-Smad3 (1:1000, abcam; cat. no. ab-40854), rabbit anti-p-Smad3 (S208) (1:1000, abcam; cat. no. ab138659), rabbit anti-Smad2/3 (1:200; Boster Biological Technology; cat. no. BA1395), rabbit anti-p-Smad2 (S465+S467)/p-Smad3 (S423+S425) (1:500; Wanlei Biological Technology, Ltd; cat. no. WL02305), rabbit anti-Smad7 (1:1000, abcam; cat. no. ab-216428), rabbit anti-NOX-4 (1:200; Boster Biological Technology; cat. no. BM4135), mouse anti-Nrf2 (1:400, Santa Cruz; cat. no. sc-81342), mouse anti Keap-1 (1:200; Santa Cruz; cat. no. sc-514914), mouse anti HO-1 (1:200; Santa Cruz; cat. no. sc-136960), mouse anti NQO-1 (1:200; Santa Cruz, Inc.; cat. no. sc-32793), rabbit β-actin (1:500; Wanlei Biological Technology, Ltd; cat. no. WL01372).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Gankyrin binds to the Kelch domain of Keap1. (A) Gankyrin influenced the binding of Keap1 to Nrf2. Equal amounts of cell lysates were immunoprecipitated with an anti-Keap1 antibody. Precipitated proteins and cell lysates were blotted with anti-Nrf2, anti-gankyrin, and anti-Keap1 antibodies. (B) Confocal microscopy was performed on HEK293T cells cotransfected with Keap1 and myc-gankyrin. Bar, 10 µm. (C and D) Gankyrin and Keap1 were cotransfected into 293T cells. Whole cell lysates were immunoprecipitated with Keap1- (C) or gankyrin-specific (D) antibodies. Precipitated proteins and cell lysates were blotted with the indicated antibodies. (E) Cell lysates from SMMC7721-con and SMMC7721-ovGank cells were immunoprecipitated with anti-Keap1 antibodies, and Western blot analysis was performed with the indicated antibodies. (F) The interaction of Myc-gankyrin with Flag-tagged truncated Keap1 fragments. The top panel shows a schematic of the truncated Keap1 fragments. HEK293T cells that were cotransfected with myc-gankyrin and Flag-tagged truncated Keap1 fragments were lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibodies. (G) The interaction of Flag-KC (Kelch domain of Keap1) with Myc-tagged gankyrin. HEK293T cells cotransfected with Flag-KC and Myc-tagged gankyrin were immunoprecipitated with anti-flag antibody and immunoblotted with anti-myc antibodies. (H) The interaction of Flag-Keap1 with Myc-tagged gankyrin mutants. The top panel shows a schematic of the gankyrin mutants. HEK293T cells cotransfected with Flag-Keap1 and myc-tagged deletion mutants of gankyrin were immunoprecipitated with anti-flag antibody. Precipitated proteins and cell lysates were blotted with anti-myc and the indicated antibodies. (I) Wild-type or ExxE motif-mutated gankyrin and Flag-Keap1 plasmids were transfected into HEK293T cells, and the cells were then lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibody. N-mutated indicated E in aa 21-24 were mutated to A, C-mutated indicated E in aa 201-204 were mutated to A, and N+C-mutated indicated E in aa 21-24 and aa 201-204 were all mutated. (J) The knockdown of Keap1 abolished the regulatory role of gankyrin on Nrf2 protein levels. Negative control oligonucleotides or siRNA targeting Keap1 were transfected into MHCCLM3-Con, -siGank, or SMMC7721-Con, -ovGank cells. Cell lysates were blotted with anti-Nrf2 and other indicated antibodies. (K) A coimmunoprecipitation assay was used to analyze the amount of gankyrin that was associated with Keap1 after stimulation with sulforaphane, tBHQ, or H 2 O 2 . SMMC7721 cells were stimulated by sulforaphane, tBHQ, or H 2 O 2 for 12 h, and the cells were then lysed and immunoprecipitated with an anti-Keap1 antibody. Precipitates and cell lysates were blotted with an anti-gankyrin antibody. The data are representative of at least two experiments with similar results.

    Journal: The Journal of Experimental Medicine

    Article Title: Gankyrin has an antioxidative role through the feedback regulation of Nrf2 in hepatocellular carcinoma

    doi: 10.1084/jem.20151208

    Figure Lengend Snippet: Gankyrin binds to the Kelch domain of Keap1. (A) Gankyrin influenced the binding of Keap1 to Nrf2. Equal amounts of cell lysates were immunoprecipitated with an anti-Keap1 antibody. Precipitated proteins and cell lysates were blotted with anti-Nrf2, anti-gankyrin, and anti-Keap1 antibodies. (B) Confocal microscopy was performed on HEK293T cells cotransfected with Keap1 and myc-gankyrin. Bar, 10 µm. (C and D) Gankyrin and Keap1 were cotransfected into 293T cells. Whole cell lysates were immunoprecipitated with Keap1- (C) or gankyrin-specific (D) antibodies. Precipitated proteins and cell lysates were blotted with the indicated antibodies. (E) Cell lysates from SMMC7721-con and SMMC7721-ovGank cells were immunoprecipitated with anti-Keap1 antibodies, and Western blot analysis was performed with the indicated antibodies. (F) The interaction of Myc-gankyrin with Flag-tagged truncated Keap1 fragments. The top panel shows a schematic of the truncated Keap1 fragments. HEK293T cells that were cotransfected with myc-gankyrin and Flag-tagged truncated Keap1 fragments were lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibodies. (G) The interaction of Flag-KC (Kelch domain of Keap1) with Myc-tagged gankyrin. HEK293T cells cotransfected with Flag-KC and Myc-tagged gankyrin were immunoprecipitated with anti-flag antibody and immunoblotted with anti-myc antibodies. (H) The interaction of Flag-Keap1 with Myc-tagged gankyrin mutants. The top panel shows a schematic of the gankyrin mutants. HEK293T cells cotransfected with Flag-Keap1 and myc-tagged deletion mutants of gankyrin were immunoprecipitated with anti-flag antibody. Precipitated proteins and cell lysates were blotted with anti-myc and the indicated antibodies. (I) Wild-type or ExxE motif-mutated gankyrin and Flag-Keap1 plasmids were transfected into HEK293T cells, and the cells were then lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibody. N-mutated indicated E in aa 21-24 were mutated to A, C-mutated indicated E in aa 201-204 were mutated to A, and N+C-mutated indicated E in aa 21-24 and aa 201-204 were all mutated. (J) The knockdown of Keap1 abolished the regulatory role of gankyrin on Nrf2 protein levels. Negative control oligonucleotides or siRNA targeting Keap1 were transfected into MHCCLM3-Con, -siGank, or SMMC7721-Con, -ovGank cells. Cell lysates were blotted with anti-Nrf2 and other indicated antibodies. (K) A coimmunoprecipitation assay was used to analyze the amount of gankyrin that was associated with Keap1 after stimulation with sulforaphane, tBHQ, or H 2 O 2 . SMMC7721 cells were stimulated by sulforaphane, tBHQ, or H 2 O 2 for 12 h, and the cells were then lysed and immunoprecipitated with an anti-Keap1 antibody. Precipitates and cell lysates were blotted with an anti-gankyrin antibody. The data are representative of at least two experiments with similar results.

    Article Snippet: Anti-PARP and anti-Keap1 were purchased from Cell Signaling Technology.

    Techniques: Binding Assay, Immunoprecipitation, Confocal Microscopy, Western Blot, Transfection, Negative Control, Co-Immunoprecipitation Assay

    Links between Nrf2 and FOXOs. Upper panel: reactive oxygen species (ROS), e.g. by interacting with Keap-1, trigger Nrf2 activation. As FOXO proteins control the expression of genes coding for antioxidant proteins, the activation of FOXO may, by blunting surges in levels of ROS, ameliorate the activity of Nrf2. Lower panel: FOXO3 was shown to also attenuate Nrf2 activity by transcriptionally upregulating the biosynthesis of Keap-1 [52] , which will bind and control Nrf2.

    Journal: Redox Biology

    Article Title: Cellular adaptation to xenobiotics: Interplay between xenosensors, reactive oxygen species and FOXO transcription factors

    doi: 10.1016/j.redox.2017.07.015

    Figure Lengend Snippet: Links between Nrf2 and FOXOs. Upper panel: reactive oxygen species (ROS), e.g. by interacting with Keap-1, trigger Nrf2 activation. As FOXO proteins control the expression of genes coding for antioxidant proteins, the activation of FOXO may, by blunting surges in levels of ROS, ameliorate the activity of Nrf2. Lower panel: FOXO3 was shown to also attenuate Nrf2 activity by transcriptionally upregulating the biosynthesis of Keap-1 [52] , which will bind and control Nrf2.

    Article Snippet: Xenobiotics that affect Keap-1/Nrf2 interaction, leading to Nrf2 activation, are usually compounds interacting with thiols, such as (i) electrophiles capable of modifying one of the Keap-1 cysteine residues, (ii) compounds metabolized to such electrophiles, (iii) certain metal(loid) ions or (iv) compounds that cause the cellular generation of reactive oxygen or nitrogen species.

    Techniques: Activation Assay, Expressing, Activity Assay

    Nrf2 activation by xenobiotics. Xenobiotics may stimulate the transcriptional activity of Nrf2 through the generation of ROS or via the formation of electrophiles. Nrf2 is controlled by binding to Keap-1, which bridges Nrf2 and Cullin-3 (Cul3), a ubiquitin ligase, thus initiating proteasomal degradation of Nrf2 and preventing its nuclear translocation. Oxidation of Keap-1 (upper right panel) by ROS may induce disulfide formation (both intra- and intermolecular) [71] , causing the release of Nrf2 from the complex, triggering Nrf2 nuclear translocation and transcriptional activation of target genes. Electrophiles may form adducts with Keap-1 through cysteine residues to elicit Nrf2 release and activation (lower right panel). The figure shows the adduct of Keap-1 generated upon reaction with the lipid peroxidation product, 4-hydroxynonenal (HNE) [72] ; different electrophiles may react with different Keap-1 cysteines, as was demonstrated for compounds such as diethyl maleate and HNE [73] , [74] .

    Journal: Redox Biology

    Article Title: Cellular adaptation to xenobiotics: Interplay between xenosensors, reactive oxygen species and FOXO transcription factors

    doi: 10.1016/j.redox.2017.07.015

    Figure Lengend Snippet: Nrf2 activation by xenobiotics. Xenobiotics may stimulate the transcriptional activity of Nrf2 through the generation of ROS or via the formation of electrophiles. Nrf2 is controlled by binding to Keap-1, which bridges Nrf2 and Cullin-3 (Cul3), a ubiquitin ligase, thus initiating proteasomal degradation of Nrf2 and preventing its nuclear translocation. Oxidation of Keap-1 (upper right panel) by ROS may induce disulfide formation (both intra- and intermolecular) [71] , causing the release of Nrf2 from the complex, triggering Nrf2 nuclear translocation and transcriptional activation of target genes. Electrophiles may form adducts with Keap-1 through cysteine residues to elicit Nrf2 release and activation (lower right panel). The figure shows the adduct of Keap-1 generated upon reaction with the lipid peroxidation product, 4-hydroxynonenal (HNE) [72] ; different electrophiles may react with different Keap-1 cysteines, as was demonstrated for compounds such as diethyl maleate and HNE [73] , [74] .

    Article Snippet: Xenobiotics that affect Keap-1/Nrf2 interaction, leading to Nrf2 activation, are usually compounds interacting with thiols, such as (i) electrophiles capable of modifying one of the Keap-1 cysteine residues, (ii) compounds metabolized to such electrophiles, (iii) certain metal(loid) ions or (iv) compounds that cause the cellular generation of reactive oxygen or nitrogen species.

    Techniques: Activation Assay, Activity Assay, Binding Assay, Translocation Assay, Generated

    Xenosensor activation by xenobiotics (Xb): simplified schematic representation. ( a,b ) Human pregnane X receptor (PXR) as well as retinoid X receptor (RXR) alpha appear to occur predominantly in the nucleus in unstimulated cultured human cells [66] , [67] , whereas constitutive androstane receptor (CAR) occurs mostly cytosolic [67] . Upon stimulation by exposure to xenobiotics (i.e. upon ligand binding), both PXR and CAR stimulate the expression of target genes by binding to their respective response elements (PXRE, PBRE) as heterodimers with RXRα. Nuclear translocation of CAR is regulated by its (de)phosphorylation: protein kinase C-dependent phosphorylation induces its cytoplasmic retention [68] , which is further supported by receptor tyrosine kinase (RTK)-dependent effects. For example, epidermal growth factor (EGF) induces, via activation of ERK, the formation of a CAR homodimer which prevents CAR dephosphorylation and thus helps retain it in the cytoplasm [69] . CAR ligands bind to the monomer, and by shifting the dimer/monomer equilibrium accordingly support CAR dephosphorylation, followed by nuclear translocation [69] . Another type of CAR activator, phenobarbital (PB) stimulates CAR indirectly, e.g. by interfering with RTK signaling: both EGF and insulin binding to their cognate receptors are attenuated in the presence of PB [30] , [31] , thus counteracting the CAR inactivation elicited by EGF or insulin. ( c ) Like PXR and CAR, peroxisome proliferator-activated receptors (PPARs) form heterodimers with RXR. RXR/PPAR heterodimers bind to PPRE (peroxisome proliferator response element) sites to stimulate transcription of target genes. Although PPARs were described as predominantly residing in the nucleus, certain stimuli were reported to elicit nuclear exclusion [70] . Moreover, nucleocytoplasmic shuttling and subcellular localization of PPARs was shown to be affected by PPAR ligands [36] . ( d ) Aryl hydrocarbon receptor (AhR) is retained in the cytoplasm in a complex containing heat shock protein (hsp) 90 as well as AhR inhibitory protein (AIP) and p23. Ligand binding releases AhR from this complex, followed by its nuclear translocation. AhR forms a heterodimer with AhR nuclear translocator (ARNT) inside the nucleus; the dimer then binds to xenobiotic response elements to stimulate transcription of target genes [44] . ( e ) Stimulation of Nrf2 transcriptional activity by xenobiotics is through interaction of xenobiotics with Keap-1, releasing Nrf2 from a complex with Keap-1 (see legend to Fig. 5 ). Following nuclear translocation, Nrf2 forms heterodimers, e.g. with small musculoaponeurotic fibrosarcoma (sMaf) proteins, that bind to antioxidant response elements (ARE) (also termed electrophile response element, EpRE) and regulate transcription [49] .

    Journal: Redox Biology

    Article Title: Cellular adaptation to xenobiotics: Interplay between xenosensors, reactive oxygen species and FOXO transcription factors

    doi: 10.1016/j.redox.2017.07.015

    Figure Lengend Snippet: Xenosensor activation by xenobiotics (Xb): simplified schematic representation. ( a,b ) Human pregnane X receptor (PXR) as well as retinoid X receptor (RXR) alpha appear to occur predominantly in the nucleus in unstimulated cultured human cells [66] , [67] , whereas constitutive androstane receptor (CAR) occurs mostly cytosolic [67] . Upon stimulation by exposure to xenobiotics (i.e. upon ligand binding), both PXR and CAR stimulate the expression of target genes by binding to their respective response elements (PXRE, PBRE) as heterodimers with RXRα. Nuclear translocation of CAR is regulated by its (de)phosphorylation: protein kinase C-dependent phosphorylation induces its cytoplasmic retention [68] , which is further supported by receptor tyrosine kinase (RTK)-dependent effects. For example, epidermal growth factor (EGF) induces, via activation of ERK, the formation of a CAR homodimer which prevents CAR dephosphorylation and thus helps retain it in the cytoplasm [69] . CAR ligands bind to the monomer, and by shifting the dimer/monomer equilibrium accordingly support CAR dephosphorylation, followed by nuclear translocation [69] . Another type of CAR activator, phenobarbital (PB) stimulates CAR indirectly, e.g. by interfering with RTK signaling: both EGF and insulin binding to their cognate receptors are attenuated in the presence of PB [30] , [31] , thus counteracting the CAR inactivation elicited by EGF or insulin. ( c ) Like PXR and CAR, peroxisome proliferator-activated receptors (PPARs) form heterodimers with RXR. RXR/PPAR heterodimers bind to PPRE (peroxisome proliferator response element) sites to stimulate transcription of target genes. Although PPARs were described as predominantly residing in the nucleus, certain stimuli were reported to elicit nuclear exclusion [70] . Moreover, nucleocytoplasmic shuttling and subcellular localization of PPARs was shown to be affected by PPAR ligands [36] . ( d ) Aryl hydrocarbon receptor (AhR) is retained in the cytoplasm in a complex containing heat shock protein (hsp) 90 as well as AhR inhibitory protein (AIP) and p23. Ligand binding releases AhR from this complex, followed by its nuclear translocation. AhR forms a heterodimer with AhR nuclear translocator (ARNT) inside the nucleus; the dimer then binds to xenobiotic response elements to stimulate transcription of target genes [44] . ( e ) Stimulation of Nrf2 transcriptional activity by xenobiotics is through interaction of xenobiotics with Keap-1, releasing Nrf2 from a complex with Keap-1 (see legend to Fig. 5 ). Following nuclear translocation, Nrf2 forms heterodimers, e.g. with small musculoaponeurotic fibrosarcoma (sMaf) proteins, that bind to antioxidant response elements (ARE) (also termed electrophile response element, EpRE) and regulate transcription [49] .

    Article Snippet: Xenobiotics that affect Keap-1/Nrf2 interaction, leading to Nrf2 activation, are usually compounds interacting with thiols, such as (i) electrophiles capable of modifying one of the Keap-1 cysteine residues, (ii) compounds metabolized to such electrophiles, (iii) certain metal(loid) ions or (iv) compounds that cause the cellular generation of reactive oxygen or nitrogen species.

    Techniques: Activation Assay, Cell Culture, Ligand Binding Assay, Expressing, Binding Assay, Translocation Assay, De-Phosphorylation Assay, Activity Assay

    NADPH-derived reactive oxygen species is required for induction of heme oxygenase-1 (HO-1) in macrophages infected with Mycobacterium tuberculosis ( Mtb ) . (A) Human monocyte-derived macrophages were infected with Mtb H37Rv [multiplicity of infection (MOI): 3] in the presence or absence of an antioxidant (Tempol) for 24 h and HO-1 levels were measured in whole cell extracts by ELISA. Right panel shows HO-1 levels in infected macrophages treated with Tempol in the presence of a TAT-conjugated NRF-2 sequence peptide that interacts with the Keap-1/NRF-2 complex (TAT-14, 50 µM). (B) Generation of superoxide anions was quantified in cell supernatants of cultures shown in panel (A) . H 2 O 2 was used as positive control. (C) Activation of the HO-1 transcription factor NRF-2 in nuclear extracts was quantitatively assessed 12 h after infection using a colorimetric DNA-binding ELISA kit. (D) Bone marrow-differentiated macrophages were prepared from wild type (WT), p47phox −/− , or gp91phox −/− (deficient in two distinct subunits of the NADPH oxidase system) mice and infected in vitro with Mtb (MOI: 3) for 24 h. HO-1 levels were then assessed in whole cell extracts by ELISA. (E,F) HO-1 levels in cell extracts as well as activation of NRF-2 in nuclear extracts were quantified in macrophage cultures in the presence of TAT-14 (50 µM). Data are from at least three experiments using cells from a total of up to six healthy donors. (D–F) Three independent experiments were performed, with samples run in triplicates. Data from different biological groups were analyzed using the Kruskal–Wallis test, with the Dunn’s multiple-comparison test, whereas matched analyses were performed using the Wilcoxon matched-pairs test (* p

    Journal: Frontiers in Immunology

    Article Title: Mycobacterium tuberculosis Induction of Heme Oxygenase-1 Expression Is Dependent on Oxidative Stress and Reflects Treatment Outcomes

    doi: 10.3389/fimmu.2017.00542

    Figure Lengend Snippet: NADPH-derived reactive oxygen species is required for induction of heme oxygenase-1 (HO-1) in macrophages infected with Mycobacterium tuberculosis ( Mtb ) . (A) Human monocyte-derived macrophages were infected with Mtb H37Rv [multiplicity of infection (MOI): 3] in the presence or absence of an antioxidant (Tempol) for 24 h and HO-1 levels were measured in whole cell extracts by ELISA. Right panel shows HO-1 levels in infected macrophages treated with Tempol in the presence of a TAT-conjugated NRF-2 sequence peptide that interacts with the Keap-1/NRF-2 complex (TAT-14, 50 µM). (B) Generation of superoxide anions was quantified in cell supernatants of cultures shown in panel (A) . H 2 O 2 was used as positive control. (C) Activation of the HO-1 transcription factor NRF-2 in nuclear extracts was quantitatively assessed 12 h after infection using a colorimetric DNA-binding ELISA kit. (D) Bone marrow-differentiated macrophages were prepared from wild type (WT), p47phox −/− , or gp91phox −/− (deficient in two distinct subunits of the NADPH oxidase system) mice and infected in vitro with Mtb (MOI: 3) for 24 h. HO-1 levels were then assessed in whole cell extracts by ELISA. (E,F) HO-1 levels in cell extracts as well as activation of NRF-2 in nuclear extracts were quantified in macrophage cultures in the presence of TAT-14 (50 µM). Data are from at least three experiments using cells from a total of up to six healthy donors. (D–F) Three independent experiments were performed, with samples run in triplicates. Data from different biological groups were analyzed using the Kruskal–Wallis test, with the Dunn’s multiple-comparison test, whereas matched analyses were performed using the Wilcoxon matched-pairs test (* p

    Article Snippet: Cells were exposed to WT H37Rv or an ESAT-6-deficient H37Rv mutant (a gift from Dr. Volker Briken, University of Maryland, College Park, MD, USA) Mtb strain at the indicated multiplicity of infection for 3 h, washed to remove extracellular bacteria, and cultured in serum-free media for 24 h in the presence or absence of the indicated concentrations of Tempol (4-hydroxy-Tempo; 4-hydroxy-2,2,6,6-tetramethylpi-peridine-N -oxyl, Sigma-Aldrich) or a TAT-conjugated NRF-2 peptide (TAT-14) that interacts with the Keap-1/NRF-2 complex (TOCRIS, Bristol, UK).

    Techniques: Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay, Sequencing, Positive Control, Activation Assay, Binding Assay, Mouse Assay, In Vitro

    ESAT6-mediated heme oxygenase-1 (HO-1) induction in Mycobacterium tuberculosis ( Mtb )-infected macrophages is associated with reactive oxygen species (ROS)-dependent release of NRF-2 to the nucleus . (A) Human monocyte-differentiated macrophages were infected with Mtb H37Rv or an H37Rv mutant strain lacking ESAT-6 expression (both at multiplicity of infection: 3) for 24 h. Cells infected with the ESAT-6-deficient Mtb strain were treated with 20 µg/mL of the fusion protein Lfn-ESAT-6 with the anthrax-protective antigen cytosolic delivery system. Generation of superoxide anions was quantified in cell supernatants using a colorimetric assay, while intracellular ROS production was assessed by flow cytometry. Results are plotted as histograms where the mean fluorescence intensity (MFI) was compared between the experimental groups. (B) Lipid peroxidation and oxidative DNA damage were assessed as described in Section “ Materials and Methods .” (C) Activation of NRF-2 in nuclear extracts (12 h postinfection) and of HO-1 levels in whole cell lysates (24 h postinfection) were determined in cultures of infected macrophages in the presence or absence of a TAT-conjugated NRF-2 sequence peptide that interacts with the Keap-1/NRF-2 complex (TAT-14, 50 µM). Data are from at least three independent experiments using cells from a total of up to six healthy donors. Data were compared using the Wilcoxon matched-pairs test (* p

    Journal: Frontiers in Immunology

    Article Title: Mycobacterium tuberculosis Induction of Heme Oxygenase-1 Expression Is Dependent on Oxidative Stress and Reflects Treatment Outcomes

    doi: 10.3389/fimmu.2017.00542

    Figure Lengend Snippet: ESAT6-mediated heme oxygenase-1 (HO-1) induction in Mycobacterium tuberculosis ( Mtb )-infected macrophages is associated with reactive oxygen species (ROS)-dependent release of NRF-2 to the nucleus . (A) Human monocyte-differentiated macrophages were infected with Mtb H37Rv or an H37Rv mutant strain lacking ESAT-6 expression (both at multiplicity of infection: 3) for 24 h. Cells infected with the ESAT-6-deficient Mtb strain were treated with 20 µg/mL of the fusion protein Lfn-ESAT-6 with the anthrax-protective antigen cytosolic delivery system. Generation of superoxide anions was quantified in cell supernatants using a colorimetric assay, while intracellular ROS production was assessed by flow cytometry. Results are plotted as histograms where the mean fluorescence intensity (MFI) was compared between the experimental groups. (B) Lipid peroxidation and oxidative DNA damage were assessed as described in Section “ Materials and Methods .” (C) Activation of NRF-2 in nuclear extracts (12 h postinfection) and of HO-1 levels in whole cell lysates (24 h postinfection) were determined in cultures of infected macrophages in the presence or absence of a TAT-conjugated NRF-2 sequence peptide that interacts with the Keap-1/NRF-2 complex (TAT-14, 50 µM). Data are from at least three independent experiments using cells from a total of up to six healthy donors. Data were compared using the Wilcoxon matched-pairs test (* p

    Article Snippet: Cells were exposed to WT H37Rv or an ESAT-6-deficient H37Rv mutant (a gift from Dr. Volker Briken, University of Maryland, College Park, MD, USA) Mtb strain at the indicated multiplicity of infection for 3 h, washed to remove extracellular bacteria, and cultured in serum-free media for 24 h in the presence or absence of the indicated concentrations of Tempol (4-hydroxy-Tempo; 4-hydroxy-2,2,6,6-tetramethylpi-peridine-N -oxyl, Sigma-Aldrich) or a TAT-conjugated NRF-2 peptide (TAT-14) that interacts with the Keap-1/NRF-2 complex (TOCRIS, Bristol, UK).

    Techniques: Infection, Mutagenesis, Expressing, Colorimetric Assay, Flow Cytometry, Cytometry, Fluorescence, Activation Assay, Sequencing

    Western blot analysis of Nrf-2 and Keap-1 protein levels in the nucleus of sham, hsham, hovx, and hovxe groups of mice. Nrf-2 level is significantly increased in the nucleus of mice treated with hyperoxia alone. Small letters denote p values. ( a p =0.008, sham vs. hsham mice), as well as in mice treated with the combination of hyperoxia and ovariectomy ( b p =0.010, sham vs. hovx) in comparison to normoxic group of animals (A). Nuclear accumulation of Keap-1 remained unchanged regardless of any treatment (B). Representative immunoblots of Nrf-2 and Keap-1 proteins in nuclear fractions of sham, hsham, hovx and hovxe group of mice are shown. Histone H3 was used to assess the purity of nuclear fraction. Amidoblack was used as a loading control. The results are presented as mean±S.D. and normalized to sham group (C). The number of samples was n =2 per each group.

    Journal: Redox Biology

    Article Title: Prominent role of exopeptidase DPP III in estrogen-mediated protection against hyperoxia in vivo

    doi: 10.1016/j.redox.2016.01.003

    Figure Lengend Snippet: Western blot analysis of Nrf-2 and Keap-1 protein levels in the nucleus of sham, hsham, hovx, and hovxe groups of mice. Nrf-2 level is significantly increased in the nucleus of mice treated with hyperoxia alone. Small letters denote p values. ( a p =0.008, sham vs. hsham mice), as well as in mice treated with the combination of hyperoxia and ovariectomy ( b p =0.010, sham vs. hovx) in comparison to normoxic group of animals (A). Nuclear accumulation of Keap-1 remained unchanged regardless of any treatment (B). Representative immunoblots of Nrf-2 and Keap-1 proteins in nuclear fractions of sham, hsham, hovx and hovxe group of mice are shown. Histone H3 was used to assess the purity of nuclear fraction. Amidoblack was used as a loading control. The results are presented as mean±S.D. and normalized to sham group (C). The number of samples was n =2 per each group.

    Article Snippet: Membranes were blocked in 5% nonfat dry milk in TN buffer (50 mM Tris–HCl, 150 mM NaCl, pH 7.4) for one hour at 37 °C, followed by overnight incubation at +4 °C with anti-DPP III polyclonal antibody (custom made by Abcore, USA), anti-Nrf2 (Abcam Inc., Cambridge, UK), diluted 1:200 and anti-Keap-1 (Abcam Inc., Cambridge, UK), diluted 1:500.

    Techniques: Western Blot, Mouse Assay

    EGb761 modulates oxidative stress response and oncogenic pathways. (A) Western blots for AKT, mTOR, ERK1/2, Nrf2 and KEAP1 in 24h, 48h and 72h treated (+) versus untreated (-) cells are shown in representative images of three independent experiments. (B) Relative expression to control (untreated cells) is demonstrated as means ± SD by quantitative analysis using densitometry normalized to the corresponding beta-actin expression; *p

    Journal: PLoS ONE

    Article Title: Ginkgo biloba induces different gene expression signatures and oncogenic pathways in malignant and non-malignant cells of the liver

    doi: 10.1371/journal.pone.0209067

    Figure Lengend Snippet: EGb761 modulates oxidative stress response and oncogenic pathways. (A) Western blots for AKT, mTOR, ERK1/2, Nrf2 and KEAP1 in 24h, 48h and 72h treated (+) versus untreated (-) cells are shown in representative images of three independent experiments. (B) Relative expression to control (untreated cells) is demonstrated as means ± SD by quantitative analysis using densitometry normalized to the corresponding beta-actin expression; *p

    Article Snippet: Membranes of full-length western blotting (WB) of (p-)AKT, (p-)mTOR, (p-)ERK1/2, Nrf2, Keap1 and corresponding beta-Actin of untreated (-) and 24h, 48h and 72h EGb761 treated (+) THLE5B and Pitts1 cells. (PDF) Click here for additional data file.

    Techniques: Western Blot, Expressing

    Keap1 ablation leads to ubiquitin aggregates accumulation and cytotoxicity. (A) Ubiquitin aggregates in Keap1 knockout cells. Keap1 +/+ and Keap1 −/− MEF cells were treated with 10 µg/ml puromycin for 4 hours, and recovered in normal

    Journal: Autophagy

    Article Title: Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy

    doi: 10.4161/auto.6.5.12189

    Figure Lengend Snippet: Keap1 ablation leads to ubiquitin aggregates accumulation and cytotoxicity. (A) Ubiquitin aggregates in Keap1 knockout cells. Keap1 +/+ and Keap1 −/− MEF cells were treated with 10 µg/ml puromycin for 4 hours, and recovered in normal

    Article Snippet: We thank Cell Signaling and Abgent Company for Keap1 antibody production.

    Techniques: Knock-Out

    Proposed model of the function of Keap1 in oxidative stress response and autophagy. In the unstressed cells, Keap1 sequesters Nrf2 in the cytosol. Keap1 is oxidized by reactive oxygen species (ROS), and Keap1 oxidation leads to Nrf2 dissociation. Nrf2

    Journal: Autophagy

    Article Title: Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy

    doi: 10.4161/auto.6.5.12189

    Figure Lengend Snippet: Proposed model of the function of Keap1 in oxidative stress response and autophagy. In the unstressed cells, Keap1 sequesters Nrf2 in the cytosol. Keap1 is oxidized by reactive oxygen species (ROS), and Keap1 oxidation leads to Nrf2 dissociation. Nrf2

    Article Snippet: We thank Cell Signaling and Abgent Company for Keap1 antibody production.

    Techniques:

    Keap1 colocalizes with LC3 and p62 in the ubiquitin aggregates. (A) Keap1 colocalizes with p62, LC3 and ubiquitin in the puromycin-treated cells. U 2 OS cells were transfected with either DsRED-Keap1 or DsRED-Keap1 together with Myc-LC3. 2 days after transfection,

    Journal: Autophagy

    Article Title: Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy

    doi: 10.4161/auto.6.5.12189

    Figure Lengend Snippet: Keap1 colocalizes with LC3 and p62 in the ubiquitin aggregates. (A) Keap1 colocalizes with p62, LC3 and ubiquitin in the puromycin-treated cells. U 2 OS cells were transfected with either DsRED-Keap1 or DsRED-Keap1 together with Myc-LC3. 2 days after transfection,

    Article Snippet: We thank Cell Signaling and Abgent Company for Keap1 antibody production.

    Techniques: Transfection

    p62 is required for Keap1 localization to ubiquitin aggregates and autophagosomes. (A) Wild-type or p62 knockout cells were transfected with DsRED-Keap1 together with Myc-LC3, followed by treatment with rapamycin (2 µM for 4 hours), cells were

    Journal: Autophagy

    Article Title: Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy

    doi: 10.4161/auto.6.5.12189

    Figure Lengend Snippet: p62 is required for Keap1 localization to ubiquitin aggregates and autophagosomes. (A) Wild-type or p62 knockout cells were transfected with DsRED-Keap1 together with Myc-LC3, followed by treatment with rapamycin (2 µM for 4 hours), cells were

    Article Snippet: We thank Cell Signaling and Abgent Company for Keap1 antibody production.

    Techniques: Knock-Out, Transfection

    Keap1 is not an autophagic substrate. (A) p62 expression was examined in wild-type or p62 knock out cells. (B) Keap1 expression was examined in wild-type or Keap1 knockout cells. (C) The protein level of Keap1, p62, tubulin and LC3 in Atg5 +/+ and Atg5

    Journal: Autophagy

    Article Title: Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy

    doi: 10.4161/auto.6.5.12189

    Figure Lengend Snippet: Keap1 is not an autophagic substrate. (A) p62 expression was examined in wild-type or p62 knock out cells. (B) Keap1 expression was examined in wild-type or Keap1 knockout cells. (C) The protein level of Keap1, p62, tubulin and LC3 in Atg5 +/+ and Atg5

    Article Snippet: We thank Cell Signaling and Abgent Company for Keap1 antibody production.

    Techniques: Expressing, Knock-Out

    Genetic ablation of Keap1 leads to defective autophagy activation. (A) Electron microscopy analysis of Keap1 +/+ and Keap1 −/− cells. Puromycin (10 µg/ml, 4 hours) treated and untreated cells were analyzed under transmission electron

    Journal: Autophagy

    Article Title: Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy

    doi: 10.4161/auto.6.5.12189

    Figure Lengend Snippet: Genetic ablation of Keap1 leads to defective autophagy activation. (A) Electron microscopy analysis of Keap1 +/+ and Keap1 −/− cells. Puromycin (10 µg/ml, 4 hours) treated and untreated cells were analyzed under transmission electron

    Article Snippet: We thank Cell Signaling and Abgent Company for Keap1 antibody production.

    Techniques: Activation Assay, Electron Microscopy, Transmission Assay

    Keap1 interacts with LC3 and p62 in a stress-inducible manner. (A) Silver staining of LC3 complex after tandem affinity purification, LC3 complex was purified from U 2 OS cell that stably expresses ZZ-FLAG-LC3 with or without starvation for one hour. Proteins’

    Journal: Autophagy

    Article Title: Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy

    doi: 10.4161/auto.6.5.12189

    Figure Lengend Snippet: Keap1 interacts with LC3 and p62 in a stress-inducible manner. (A) Silver staining of LC3 complex after tandem affinity purification, LC3 complex was purified from U 2 OS cell that stably expresses ZZ-FLAG-LC3 with or without starvation for one hour. Proteins’

    Article Snippet: We thank Cell Signaling and Abgent Company for Keap1 antibody production.

    Techniques: Silver Staining, Affinity Purification, Purification, Stable Transfection

    Keap1 levels are unaltered while the concentration of Cullin-3 is decreased in EAE. (a) Keap1 protein levels in the spinal cord of control and EAE mice were determined by western blot analysis as described under “Materials and Methods”, and were normalized by the amount of coomassie blue stain in the same gel lane. Values represent the mean ± SEM of 3 animals per experimental group. Inset panel shows a representative western blot. (b) Cullin-3 levels in the spinal cord of control and EAE mice at 21 dpi were determined by western blot analysis as described under “Materials and Methods” and are expressed relative to those of GAPDH. Values represent the mean ± SEM of 3 animals per group. Asterisks denote values that are statistically different ( p

    Journal: Journal of neurochemistry

    Article Title: Nrf2-dysregulation correlates with reduced synthesis and low glutathione levels in experimental autoimmune encephalomyelitis

    doi: 10.1111/jnc.13837

    Figure Lengend Snippet: Keap1 levels are unaltered while the concentration of Cullin-3 is decreased in EAE. (a) Keap1 protein levels in the spinal cord of control and EAE mice were determined by western blot analysis as described under “Materials and Methods”, and were normalized by the amount of coomassie blue stain in the same gel lane. Values represent the mean ± SEM of 3 animals per experimental group. Inset panel shows a representative western blot. (b) Cullin-3 levels in the spinal cord of control and EAE mice at 21 dpi were determined by western blot analysis as described under “Materials and Methods” and are expressed relative to those of GAPDH. Values represent the mean ± SEM of 3 animals per group. Asterisks denote values that are statistically different ( p

    Article Snippet: [ ] Lo SC, Hannink M. PGAM5 tethers a ternary complex containing Keap1 and Nrf2 to mitochondria.

    Techniques: Concentration Assay, Mouse Assay, Western Blot, Staining

    The restorative effect of mitoQ on PINK and Parkin expression was mediated in part by Nrf2/Keap1. A: Immunoblotting assay for Keap1 and Nrf2 expression, which showed that mitoQ inhibited Keap1 and cytoplasmic Nrf2 expression in HK-2 cells exposed to HG and restored nuclear Nrf2 expression. A1-A3: Quantification of average Western blot band intensity. B: Immunofluorescence assay for Nrf2 showing decreased antibody staining intensity in HK-2 cells after incubation under HG conditions. MitoQ up-regulated the inhibition of Nrf2 intensity in HG-exposed cells. C: Bar graphs depicting Nrf2 binding to the ARE. D: The interaction between Keap1 and Nrf2 in HK-2 cells subjected to HG exposure with or without mitoQ was assessed using IP. D1 and D2: Quantification of the intensity of the interaction between Keap1 and Nrf2 using IP. E: Western blot analysis revealed that mitoQ restored PINK and Parkin expression in HK-2 cells exposed to HG, an effect that was partially abolished by transfection with Keap1 siRNA or Nrf2 siRNA. E1: Quantification of average Western blot band intensity. F: Similar results were observed for PINK mRNA expression, as demonstrated by real-time PCR. *P

    Journal: Redox Biology

    Article Title: The mitochondria-targeted antioxidant MitoQ ameliorated tubular injury mediated by mitophagy in diabetic kidney disease via Nrf2/PINK1

    doi: 10.1016/j.redox.2016.12.022

    Figure Lengend Snippet: The restorative effect of mitoQ on PINK and Parkin expression was mediated in part by Nrf2/Keap1. A: Immunoblotting assay for Keap1 and Nrf2 expression, which showed that mitoQ inhibited Keap1 and cytoplasmic Nrf2 expression in HK-2 cells exposed to HG and restored nuclear Nrf2 expression. A1-A3: Quantification of average Western blot band intensity. B: Immunofluorescence assay for Nrf2 showing decreased antibody staining intensity in HK-2 cells after incubation under HG conditions. MitoQ up-regulated the inhibition of Nrf2 intensity in HG-exposed cells. C: Bar graphs depicting Nrf2 binding to the ARE. D: The interaction between Keap1 and Nrf2 in HK-2 cells subjected to HG exposure with or without mitoQ was assessed using IP. D1 and D2: Quantification of the intensity of the interaction between Keap1 and Nrf2 using IP. E: Western blot analysis revealed that mitoQ restored PINK and Parkin expression in HK-2 cells exposed to HG, an effect that was partially abolished by transfection with Keap1 siRNA or Nrf2 siRNA. E1: Quantification of average Western blot band intensity. F: Similar results were observed for PINK mRNA expression, as demonstrated by real-time PCR. *P

    Article Snippet: Notably, immunoprecipitation demonstrated an interaction between Nrf2 and Keap1 in HK-2 cells under basal conditions.

    Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Incubation, Inhibition, Binding Assay, Transfection, Real-time Polymerase Chain Reaction

    The effect of mitoQ on the expression of the mitophagy-associated proteins PINK and Parkin and the oxidative stress adaptors Keap1 and Nrf2 in db/db mice. A: IHC, kidney sections in db/m mice, db/db mice and db/db mice treated with mitoQ were stained with Keap1 (column 1), Nrf2 (column2), PINK (column3) and Parkin (column4) antibodies. PINK, Parkin, and Nrf2 intensity decreased in the tubules in the kidneys of db/db mice compared to those of control mice (middle panels vs. top panels). These intensities were restored by mitoQ treatment (bottom panels vs. middle panels). Conversely, Keap1 intensity was increased in db/db mice, but this increase was partially inhibited after mitoQ treatment. B1-B4: Semiquantification of IHC staining of Keap1, Nrf2, PINK and Parkin in kidney tissues. C: Western blot analysis of Keap1, Nrf2, PINK, Parkin and phosphorylated-Parkin ( p -Parkin) expression. D1-D5: Quantification of average Western blot band intensity. Values are the mean±SE. *P

    Journal: Redox Biology

    Article Title: The mitochondria-targeted antioxidant MitoQ ameliorated tubular injury mediated by mitophagy in diabetic kidney disease via Nrf2/PINK1

    doi: 10.1016/j.redox.2016.12.022

    Figure Lengend Snippet: The effect of mitoQ on the expression of the mitophagy-associated proteins PINK and Parkin and the oxidative stress adaptors Keap1 and Nrf2 in db/db mice. A: IHC, kidney sections in db/m mice, db/db mice and db/db mice treated with mitoQ were stained with Keap1 (column 1), Nrf2 (column2), PINK (column3) and Parkin (column4) antibodies. PINK, Parkin, and Nrf2 intensity decreased in the tubules in the kidneys of db/db mice compared to those of control mice (middle panels vs. top panels). These intensities were restored by mitoQ treatment (bottom panels vs. middle panels). Conversely, Keap1 intensity was increased in db/db mice, but this increase was partially inhibited after mitoQ treatment. B1-B4: Semiquantification of IHC staining of Keap1, Nrf2, PINK and Parkin in kidney tissues. C: Western blot analysis of Keap1, Nrf2, PINK, Parkin and phosphorylated-Parkin ( p -Parkin) expression. D1-D5: Quantification of average Western blot band intensity. Values are the mean±SE. *P

    Article Snippet: Notably, immunoprecipitation demonstrated an interaction between Nrf2 and Keap1 in HK-2 cells under basal conditions.

    Techniques: Expressing, Mouse Assay, Immunohistochemistry, Staining, Western Blot

    Schematic diagram depicting the possible molecular mechanisms by which mitoQ prevents renal tubular cell injury by maintaining mitochondrial integrity under HG conditions. Under HG conditions, nuclear Nrf2 expression and activity are down-regulated in tubular cells, which decreases PINK1 transcription and subsequent Parkin phosphorylations and then leads to defective mitophagy. This insufficient mitophagy results in ROS overproduction and aberrant mitochondrial dynamics characterized by Drp1 activation and Mfn2 suppression, changes accompanied by mitochondrial dysfunction, fragmented mitochondria accumulation and mitochondrial apoptotic pathway activation, i.e., caspase 3 release, which eventually leads to cell apoptosis. Interestingly, mitoQ treatment increases the dissociation of Nrf2 from Keap1, and the nuclear location of the former up-regulates the transcription of PINK and restores mitophagy, which maintains mitochondrial quality control, thereby attenuating hyperglycemia-induced tubular injury and apoptosis.

    Journal: Redox Biology

    Article Title: The mitochondria-targeted antioxidant MitoQ ameliorated tubular injury mediated by mitophagy in diabetic kidney disease via Nrf2/PINK1

    doi: 10.1016/j.redox.2016.12.022

    Figure Lengend Snippet: Schematic diagram depicting the possible molecular mechanisms by which mitoQ prevents renal tubular cell injury by maintaining mitochondrial integrity under HG conditions. Under HG conditions, nuclear Nrf2 expression and activity are down-regulated in tubular cells, which decreases PINK1 transcription and subsequent Parkin phosphorylations and then leads to defective mitophagy. This insufficient mitophagy results in ROS overproduction and aberrant mitochondrial dynamics characterized by Drp1 activation and Mfn2 suppression, changes accompanied by mitochondrial dysfunction, fragmented mitochondria accumulation and mitochondrial apoptotic pathway activation, i.e., caspase 3 release, which eventually leads to cell apoptosis. Interestingly, mitoQ treatment increases the dissociation of Nrf2 from Keap1, and the nuclear location of the former up-regulates the transcription of PINK and restores mitophagy, which maintains mitochondrial quality control, thereby attenuating hyperglycemia-induced tubular injury and apoptosis.

    Article Snippet: Notably, immunoprecipitation demonstrated an interaction between Nrf2 and Keap1 in HK-2 cells under basal conditions.

    Techniques: Expressing, Activity Assay, Activation Assay