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  • 94
    Thermo Fisher gene exp keap1 hs00202227 m1
    Gene Exp Keap1 Hs00202227 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti keap1 antibody
    Systemic NRF2 activation by <t>Keap1</t> knockdown alleviates tissue inflammation in Sf mice. (A) Macroscopic observation of the pinnae and tails of WT, Sf, and Sf:: Keap1 FA/− mice. (B) HE staining of the skin (pinnae), livers, and lungs of WT, Sf, and Sf:: Keap1 FA/− mice. Arrows indicate leukocyte infiltration. (C) Survival curves for WT ( n = 30), Sf ( n = 26), and Sf:: Keap1 FA/− ( n = 17) mice. (D) Macroscopic observation of WT, Sf, and Sf:: Keap1 FA/− mice. (E) Body weight measurements of WT ( n = 10), Sf ( n = 10), and Sf:: Keap1 FA/− ( n = 10) mice at 14 days of age. The values represent the means and SD. *, P
    Anti Keap1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc keap1
    PHL targeted <t>Keap1</t> to disassemble the Keap1/Nrf2 complex. (A) Per-residue of top 10 contribution to the binding free energy; (B) Structural analysis of the most 10 contributors of Keap1 to PHL, hydrogen bonds are colored yellow; (C) Alignment of the representative structures between from conventional MD simulation and from GaMD simulation; (D) PCA scatter plot of 200,000 snapshots from GaMD simulation along the first two principal components. (E) Immunoblotting analysis of Nrf2 expression and Co-Immunoprecipitation analysis of Nrf2 and Keap1 complex in lysates prepared from heart tissues. GAPDH was used as a loading control.
    Keap1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 642 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology keap1
    <t>Keap1,</t> MCM3, and MCM-BP form a ternary complex. ( a ) Strep-Keap1 and FLAG-MCM3 pulldown experiments from Sf9 cells co-infected with baculoviruses expressing mouse MCM-BP together with WT or interaction deficient mutant MCM3 and Keap1 as indicated. Top panels show the Western blots of indicated proteins, bottom panel the blotted membranes that were stained with colloidal gold total protein stain. 1/300th of the starting extracts (‘input’) and 1/6th of the pulldown samples was loaded on each lane. See Supplementary Fig. S6 for full-length blots. ( b ) Strep-Keap1 - FLAG-MCM3 tandem affinity purification experiment from Sf9 cells co-infected with baculoviruses expressing all six mouse MCM2-7 subunits, Keap1, and MCM-BP. Coomassie brilliant blue stained SDS-PAGE gel on the left shows eluted material from both affinity purification steps, and unbound material from the FLAG affinity step in the middle lane. Resulting complexes were further resolved by Superose 6 size exclusion chromatography, the fractions of which are shown on right gel; co-elution of molecular weight markers is indicated at the bottom. The identity of protein bands was verified by mass spectrometry.
    Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 791 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    keap1  (Abcam)
    92
    Abcam keap1
    miR-29 regulates <t>Keap1</t> expression in HK-2 cells. a Cells were transfected with miR-29 mimic and b miR-29 inhibitor as well as respective negative control (NC) for 48 h; miR-29 level, translational activity of Keap1 and expression of Keap1 mRNA and protein were determined. Data were presented as mean ± S.D. ** P
    Keap1, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteintech rabbit keap1 polyclonal
    Nrf2 and <t>Keap1</t> immunohistochemical stains in normal esophageal mucosae and esophageal squamous cell carcinoma (ESCC). ( a ) Representative cases of Nrf2 staining. Neither cytoplasmic nor nuclear expression of Nrf2 was common in normal esophageal mucosa (intensity = 0). ESCC samples displayed increased Nrf2 staining in both the cytoplasm and nucleus. ( b ) Representative cases of Keap1 staining. Keap1 expression was high in the normal esophageal mucosa (intensity = 3). ESCC showed various staining patterns of Keap1 and limited expression was common. (Original magnification = 400,Scale bar 50 μm). Black arrows indicate positive nuclear staining and white arrows indicate positive cytoplasmic staining. ( c ) Comparison of immunohistochemical Q scores of Nrf2 and Keap1 between ESCC and normal esophageal mucosae. The medium lines of boxes show the median value, the top and bottom lines of boxes represent the 75th and 25th percentiles, respectively; and the ends of whiskers represent the 10th and 90th percentiles.
    Rabbit Keap1 Polyclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 99/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Novoprotein keap1
    Manipulation of SQSTM1 levels affects the basal protein level of <t>Keap1</t> in a panel of human cell lines. A , HeLa, HEK293T, HepG2, Huh-7 and CFPAC-1 cells were transfected with a scrambled control siRNA (si-CON) or SQSTM1-targeting siRNA (si-SQSTM1 h3) for 48 h. Keap1 and SQSTM1 levels were analyzed by Western blotting. Immunoreactive band volumes were quantified by densitometry and expressed relative to β-actin to enable comparison of Keap1 protein levels in cells transfected with si-CON, which were arbitrarily set at 1.00, and those transfected with si-SQSTM1 h3. Relative Keap1 levels are presented beneath the Keap1 blot. B , HeLa, HEK293T, and HepG2 cells were mock transfected or transfected with a SQSTM1-FLAG/His construct for 48 h. Keap1 and SQSTM1 levels were analyzed by Western blotting. Immunoreactive band volumes were quantified by densitometry and expressed relative to β-actin to enable comparison of Keap1 protein levels in mock transfected cells, which were arbitrarily set at 1.00, and those transfected with SQSTM1-FLAG/His. Relative Keap1 levels are presented beneath the Keap1 blot.
    Keap1, supplied by Novoprotein, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology anti keap1
    Silencing of <t>Keap1</t> in the liver by siRNA administration in vivo reduces ConA-mediated apoptosis. Analysis of cell-death markers in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A) TNFα mRNA levels determined by real-time PCR. Data are presented as mean±s.e.m. relative to siLuc mice. (B) Representative blots with the indicated antibodies. (C) Graphics of caspase 3 and 8 enzymatic activities. Data are presented as mean±s.e.m. relative to siLuc mice. (D) Representative blots with the indicated antibodies. * P
    Anti Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    OriGene keap1
    Effect of SKI-II on <t>Keap1.</t> A. Nuclear fractions from BEAS2B cells treated with SKI-II (1 µM) at increasing times (10–120 min) were analysed for Keap1 and Nrf2 expression and normalized using TBP (nuclear). Keap1 bands at 140 kDa and 69 kDa were analysed as fold change over non-treatment of the 69 kDa band. *** p
    Keap1, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti keap1
    Working model for the inhibitory of NOM on IL‐1β–induced inflammation and ECM degradation in mice chondrocytes by targeting the <t>Keap1‐Nrf2</t> signalling. ECM, extracellular matrix; HO‐1, haem oxygenase‐1; IL, interleukin; Keap1, Kelch‐like ECH‐associated protein 1; NF‐κB, nuclear factor κB; NOM, nomilin; Nrf2, nuclear factor‐erythroid 2‐related factor‐2; OA, osteoarthritis
    Anti Keap1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher keap1
    Working model for the inhibitory of NOM on IL‐1β–induced inflammation and ECM degradation in mice chondrocytes by targeting the <t>Keap1‐Nrf2</t> signalling. ECM, extracellular matrix; HO‐1, haem oxygenase‐1; IL, interleukin; Keap1, Kelch‐like ECH‐associated protein 1; NF‐κB, nuclear factor κB; NOM, nomilin; Nrf2, nuclear factor‐erythroid 2‐related factor‐2; OA, osteoarthritis
    Keap1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp keap1 mm00497268 m1
    The messenger RNA (mRNA) levels of nuclear factor erythroid-2 like 2 (NRF2), heme-oxygenase1 (HMOX1), and kelch-like ECH-associated protein 1 <t>(KEAP1)</t> of diaphragm muscle. Data are expressed as mean ± standard deviation. A = p
    Gene Exp Keap1 Mm00497268 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Addgene inc flag keap1
    Functional Consequences of <t>E1-Keap1</t> Interaction ( a ) Subnetwork of interaction between HPV E1 and Keap1. Blue arrows denote the main contribution of differential mutation scores to KEAP1 through network propagation. MiST score of virus-human interaction in C33A cells is indicated (0.81). The mechanism of interaction is represented by edge shapes, with +Ub indicating ubiquitination. ( b , c , d , e ) Western Blot analysis of virus-host interaction by immunoprecipitation (IP) using streptavidin-coated beads to bind the Strep-tag ( b , d , e ) or an antibody against endogenous Keap1 ( c ). Proteins indicated on the right of each blot from IP and input samples were detected using the antibodies indicated. Bands were cropped from the same original membrane. Distinction between different strains of HPV is made using strain number preceding the respective viral bait (HPV-31 = 31, HPV-16 = 16; d ). ( f ) Cartoons depicting the truncation / deletion mutants of HPV-31 E1. Scale is provided in amino acid numbers of the full-length protein at the top. ( g ) Luciferase reporter assay for the antioxidant response element (ARE). Relative luciferase light units were normalized based on transfection control. The mean ± standard deviation of technical triplicates are depicted. ( h ) Model depicting how E1 influences the Keap1-Nrf2 pathway.
    Flag Keap1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology goat anti keap1
    <t>Keap1</t> knockdown sensitizes liver cells to PA-induced toxicity, and overexpression of Keap1 mutant (Keap1 ΔCTR) protects against lipotoxicity. ( a and b ) shKeap1#4 and shLuc Hep3B cells were treated with PA at 400 μ M or vehicle (V) for 6 h. ( a ) Caspase 3/7 catalytic activity was measured using a fluorogenic assay. ( b ) Cell death was determined by trypan blue exclusion assay. ( c ) Whole-cell lysates were prepared from shKeap1#4 and shLuc Hep3B cells treated with PA at 400 or 800 μ M or vehicle (V) for 6 h. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( d ) Whole-cell lysates were prepared from WT or hepatocyte-specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes. Immunoblot analysis were performed for mKeap1, mNrf2 and β -actin. ( e ) Isolated WT or Keap1 −/− HKO primary mouse hepatocytes were treated for 24 h with PA at 400 μ M or vehicle, and apoptotic nuclei were counted after DAPI staining. ( f ) Whole-cell lysates were prepared from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated at the indicated time points with PA 400 μ M or vehicle. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and β -actin. ( g ) Cell death was determined by trypan blue exclusion assay in Keap1 ΔCTR or control Hep3B cells treated with PA at 400 μ M or vehicle for 16 h. All data are expressed as mean±S.E.M. for three experiments; *P
    Goat Anti Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Proteintech mouse keap1 monoclonal
    BMSC multipotency dependent on restoration of Sox2 expression. A : Relative expression of Sox2. B : Time course of relative expression of Sox2 and YAP after <t>Keap1</t> knockdown. * P
    Mouse Keap1 Monoclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech keap1
    <t>Keap1</t> ΔC competes against effects of Keap1 on the Nrf2-mediated reporter gene. ( A ) Three distinct dimers of Keap1 and Keap1 ΔC with different views were simulated by the VMD1.9.3 software, based on a template of the crystal structure of Keap1 (3WN7 deposited in PDB). ( B ) Shows schematic of the structural domains of Keap1, Keap1 ΔC , as well as other mutants Keap1 N321 and Keap1 ΔN , some of which were tagged by the V5 ectope at the N-terminal or C-terminal ends. ( C , D ) HepG2 cells were co-transfected with an Nrf2 expression construct, and both reporters of pARE-Luc and pRL-TK (as an internal control), together with Keap1, Keap1 ΔC and other mutants as indicated. After 24 h, luciferase activity was measured with significant changes ($ p
    Keap1, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    R&D Systems human mouse rat keap1 antibody
    <t>Keap1</t> ΔC competes against effects of Keap1 on the Nrf2-mediated reporter gene. ( A ) Three distinct dimers of Keap1 and Keap1 ΔC with different views were simulated by the VMD1.9.3 software, based on a template of the crystal structure of Keap1 (3WN7 deposited in PDB). ( B ) Shows schematic of the structural domains of Keap1, Keap1 ΔC , as well as other mutants Keap1 N321 and Keap1 ΔN , some of which were tagged by the V5 ectope at the N-terminal or C-terminal ends. ( C , D ) HepG2 cells were co-transfected with an Nrf2 expression construct, and both reporters of pARE-Luc and pRL-TK (as an internal control), together with Keap1, Keap1 ΔC and other mutants as indicated. After 24 h, luciferase activity was measured with significant changes ($ p
    Human Mouse Rat Keap1 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Systemic NRF2 activation by Keap1 knockdown alleviates tissue inflammation in Sf mice. (A) Macroscopic observation of the pinnae and tails of WT, Sf, and Sf:: Keap1 FA/− mice. (B) HE staining of the skin (pinnae), livers, and lungs of WT, Sf, and Sf:: Keap1 FA/− mice. Arrows indicate leukocyte infiltration. (C) Survival curves for WT ( n = 30), Sf ( n = 26), and Sf:: Keap1 FA/− ( n = 17) mice. (D) Macroscopic observation of WT, Sf, and Sf:: Keap1 FA/− mice. (E) Body weight measurements of WT ( n = 10), Sf ( n = 10), and Sf:: Keap1 FA/− ( n = 10) mice at 14 days of age. The values represent the means and SD. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Systemic Activation of NRF2 Alleviates Lethal Autoimmune Inflammation in Scurfy Mice

    doi: 10.1128/MCB.00063-17

    Figure Lengend Snippet: Systemic NRF2 activation by Keap1 knockdown alleviates tissue inflammation in Sf mice. (A) Macroscopic observation of the pinnae and tails of WT, Sf, and Sf:: Keap1 FA/− mice. (B) HE staining of the skin (pinnae), livers, and lungs of WT, Sf, and Sf:: Keap1 FA/− mice. Arrows indicate leukocyte infiltration. (C) Survival curves for WT ( n = 30), Sf ( n = 26), and Sf:: Keap1 FA/− ( n = 17) mice. (D) Macroscopic observation of WT, Sf, and Sf:: Keap1 FA/− mice. (E) Body weight measurements of WT ( n = 10), Sf ( n = 10), and Sf:: Keap1 FA/− ( n = 10) mice at 14 days of age. The values represent the means and SD. *, P

    Article Snippet: The samples were then subjected to immunoblot analysis using an anti-NRF2 antibody (clone 103; 1:500) , an anti-KEAP1 antibody (clone 111; 1:200) , and an anti-α-tubulin antibody (clone DM1A; 1:10,000) (Sigma-Aldrich).

    Techniques: Activation Assay, Mouse Assay, Staining

    T cell-specific Keap1 deletion does not alter phenotypic activation of T cells in Sf mice. (A) Frequencies of T cell populations in the LNs of control ( n = 4), Sf::Control ( n = 6), and Sf:: Keap1 -TKO ( n = 3) mice. Percentages of CD4 and CD8 single-positive cells in LN cells (left) and percentages of CD25 + , CD44 + , and CD69 + cells in CD4 single-positive cells (right) are shown. The values indicate the means and SD. (B) Representative histogram data for CD4 single-positive LN cells from control, Sf::Control, and Sf:: Keap1 -TKO mice, with percentages of CD25 + , CD44 + , and CD69 + cells. NS, not significant; *, P

    Journal: Molecular and Cellular Biology

    Article Title: Systemic Activation of NRF2 Alleviates Lethal Autoimmune Inflammation in Scurfy Mice

    doi: 10.1128/MCB.00063-17

    Figure Lengend Snippet: T cell-specific Keap1 deletion does not alter phenotypic activation of T cells in Sf mice. (A) Frequencies of T cell populations in the LNs of control ( n = 4), Sf::Control ( n = 6), and Sf:: Keap1 -TKO ( n = 3) mice. Percentages of CD4 and CD8 single-positive cells in LN cells (left) and percentages of CD25 + , CD44 + , and CD69 + cells in CD4 single-positive cells (right) are shown. The values indicate the means and SD. (B) Representative histogram data for CD4 single-positive LN cells from control, Sf::Control, and Sf:: Keap1 -TKO mice, with percentages of CD25 + , CD44 + , and CD69 + cells. NS, not significant; *, P

    Article Snippet: The samples were then subjected to immunoblot analysis using an anti-NRF2 antibody (clone 103; 1:500) , an anti-KEAP1 antibody (clone 111; 1:200) , and an anti-α-tubulin antibody (clone DM1A; 1:10,000) (Sigma-Aldrich).

    Techniques: Activation Assay, Mouse Assay

    Systemic NRF2 activation inhibits cytokine production of T cells in Sf mice. (A) Frequencies of effector T cell subsets in the LNs of WT (for IL-10 + cells, n = 4; for others, n = 7), Sf (for IL-10 + cells, n = 3; for others, n = 6), and Sf:: Keap1 F/− (for IL-10 + cells, n = 4; for others, n = 8) mice. Percentages of the IFN-γ + , IL-4 + , IL-17A + , and IL-10 + fractions of CD4 single-positive cells and the percentage of the IFN-γ + fraction of CD8 single-positive cells are shown. The values represent the means and SD. (B) Representative contour plot data for the IFN-γ + , IL-4 + , IL-17A + , and IL-10 + fractions of CD4 single-positive LN cells and the IFN-γ + fraction of CD8 single-positive LN cells, with the percentages of cytokine-positive fractions shown. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Systemic Activation of NRF2 Alleviates Lethal Autoimmune Inflammation in Scurfy Mice

    doi: 10.1128/MCB.00063-17

    Figure Lengend Snippet: Systemic NRF2 activation inhibits cytokine production of T cells in Sf mice. (A) Frequencies of effector T cell subsets in the LNs of WT (for IL-10 + cells, n = 4; for others, n = 7), Sf (for IL-10 + cells, n = 3; for others, n = 6), and Sf:: Keap1 F/− (for IL-10 + cells, n = 4; for others, n = 8) mice. Percentages of the IFN-γ + , IL-4 + , IL-17A + , and IL-10 + fractions of CD4 single-positive cells and the percentage of the IFN-γ + fraction of CD8 single-positive cells are shown. The values represent the means and SD. (B) Representative contour plot data for the IFN-γ + , IL-4 + , IL-17A + , and IL-10 + fractions of CD4 single-positive LN cells and the IFN-γ + fraction of CD8 single-positive LN cells, with the percentages of cytokine-positive fractions shown. *, P

    Article Snippet: The samples were then subjected to immunoblot analysis using an anti-NRF2 antibody (clone 103; 1:500) , an anti-KEAP1 antibody (clone 111; 1:200) , and an anti-α-tubulin antibody (clone DM1A; 1:10,000) (Sigma-Aldrich).

    Techniques: Activation Assay, Mouse Assay

    Dendritic cell- or myeloid cell-specific Keap1 deletion does not ameliorate the autoimmune response in Sf mice. (A and C) Macroscopic observations of Sf:: Keap1 -MKO (A) and Sf:: Keap1 -DKO (C) mice compared to control and Sf::Control mice. (B and D) Frequencies of T cell populations in the LNs of Sf:: Keap1 -MKO ( n = 5) (B) and Sf:: Keap1 -DKO ( n = 4) (D) mice compared to control ( n = 6 for panel B; n = 4 for panel D) and Sf::Control ( n = 7 for panel B; n = 4 for panel D) mice. Percentages of CD4 and CD8 single-positive cells in LN cells (left) and percentages of CD25 + , CD44 + , and CD69 + cells in CD4 single-positive cells (right) are shown. The values indicate the means and SD. NS, not significant; *, P

    Journal: Molecular and Cellular Biology

    Article Title: Systemic Activation of NRF2 Alleviates Lethal Autoimmune Inflammation in Scurfy Mice

    doi: 10.1128/MCB.00063-17

    Figure Lengend Snippet: Dendritic cell- or myeloid cell-specific Keap1 deletion does not ameliorate the autoimmune response in Sf mice. (A and C) Macroscopic observations of Sf:: Keap1 -MKO (A) and Sf:: Keap1 -DKO (C) mice compared to control and Sf::Control mice. (B and D) Frequencies of T cell populations in the LNs of Sf:: Keap1 -MKO ( n = 5) (B) and Sf:: Keap1 -DKO ( n = 4) (D) mice compared to control ( n = 6 for panel B; n = 4 for panel D) and Sf::Control ( n = 7 for panel B; n = 4 for panel D) mice. Percentages of CD4 and CD8 single-positive cells in LN cells (left) and percentages of CD25 + , CD44 + , and CD69 + cells in CD4 single-positive cells (right) are shown. The values indicate the means and SD. NS, not significant; *, P

    Article Snippet: The samples were then subjected to immunoblot analysis using an anti-NRF2 antibody (clone 103; 1:500) , an anti-KEAP1 antibody (clone 111; 1:200) , and an anti-α-tubulin antibody (clone DM1A; 1:10,000) (Sigma-Aldrich).

    Techniques: Mouse Assay

    Systemic NRF2 activation inhibits the activation of T cells in Sf mice. (A) Ratios of the weights of the spleens and LNs (bilateral axillary, brachial, and inguinal LNs) to the whole-body weights of WT ( n = 10), Sf ( n = 10), and Sf:: Keap1 FA/− ( n = 10) mice at 17 days of age. The values represent the means and SD. (B) Macroscopic observation of the spleens and LNs of WT, Sf, and Sf:: Keap1 FA/− mice. (C) Frequencies of T cell populations in the LNs of WT ( n = 5), Sf ( n = 4), and Sf:: Keap1 F/− ( n = 8) mice. Percentages of CD4 and CD8 single-positive cells in LN cells (left) and percentages of CD25 + , CD44 + , and CD69 + cells in CD4 single-positive cells (right) are shown. The values indicate the means and SD. (D) Representative histogram data for CD4 single-positive LN cells from WT, Sf, and Sf:: Keap1 FA/− mice at 15 to 19 days of age, with percentages of CD25 + , CD44 + , and CD69 + cells. NS, not significant; *, P

    Journal: Molecular and Cellular Biology

    Article Title: Systemic Activation of NRF2 Alleviates Lethal Autoimmune Inflammation in Scurfy Mice

    doi: 10.1128/MCB.00063-17

    Figure Lengend Snippet: Systemic NRF2 activation inhibits the activation of T cells in Sf mice. (A) Ratios of the weights of the spleens and LNs (bilateral axillary, brachial, and inguinal LNs) to the whole-body weights of WT ( n = 10), Sf ( n = 10), and Sf:: Keap1 FA/− ( n = 10) mice at 17 days of age. The values represent the means and SD. (B) Macroscopic observation of the spleens and LNs of WT, Sf, and Sf:: Keap1 FA/− mice. (C) Frequencies of T cell populations in the LNs of WT ( n = 5), Sf ( n = 4), and Sf:: Keap1 F/− ( n = 8) mice. Percentages of CD4 and CD8 single-positive cells in LN cells (left) and percentages of CD25 + , CD44 + , and CD69 + cells in CD4 single-positive cells (right) are shown. The values indicate the means and SD. (D) Representative histogram data for CD4 single-positive LN cells from WT, Sf, and Sf:: Keap1 FA/− mice at 15 to 19 days of age, with percentages of CD25 + , CD44 + , and CD69 + cells. NS, not significant; *, P

    Article Snippet: The samples were then subjected to immunoblot analysis using an anti-NRF2 antibody (clone 103; 1:500) , an anti-KEAP1 antibody (clone 111; 1:200) , and an anti-α-tubulin antibody (clone DM1A; 1:10,000) (Sigma-Aldrich).

    Techniques: Activation Assay, Mouse Assay

    T cell-specific Keap1 deletion alleviates skin inflammation. (A) Gene expression of Keap1 and Nqo1 in T cells (CD3e + ) and the rest of the cells (CD3e − ) from the spleens of control ( n = 3) and Sf:: Keap1 -TKO ( n = 3) mice. The expression levels were normalized to Gapdh expression, and the expression level in control mice was set to 1. The values represent the means and SD. (B) Macroscopic observation of the pinnae and tails of control, Sf::Control, and Sf:: Keap1 -TKO mice. (C) HE staining of the skin (pinnae), livers, and lungs of control, Sf::Control, and Sf:: Keap1 -TKO mice. Arrows indicate leukocyte infiltration. NS, not significant; *, P

    Journal: Molecular and Cellular Biology

    Article Title: Systemic Activation of NRF2 Alleviates Lethal Autoimmune Inflammation in Scurfy Mice

    doi: 10.1128/MCB.00063-17

    Figure Lengend Snippet: T cell-specific Keap1 deletion alleviates skin inflammation. (A) Gene expression of Keap1 and Nqo1 in T cells (CD3e + ) and the rest of the cells (CD3e − ) from the spleens of control ( n = 3) and Sf:: Keap1 -TKO ( n = 3) mice. The expression levels were normalized to Gapdh expression, and the expression level in control mice was set to 1. The values represent the means and SD. (B) Macroscopic observation of the pinnae and tails of control, Sf::Control, and Sf:: Keap1 -TKO mice. (C) HE staining of the skin (pinnae), livers, and lungs of control, Sf::Control, and Sf:: Keap1 -TKO mice. Arrows indicate leukocyte infiltration. NS, not significant; *, P

    Article Snippet: The samples were then subjected to immunoblot analysis using an anti-NRF2 antibody (clone 103; 1:500) , an anti-KEAP1 antibody (clone 111; 1:200) , and an anti-α-tubulin antibody (clone DM1A; 1:10,000) (Sigma-Aldrich).

    Techniques: Expressing, Mouse Assay, Staining

    Production of IFN-γ is dramatically reduced in Sf mice in the Keap1 knockdown background. (A) Serum cytokine levels in WT ( n = 10), Keap1 FA/− ( n = 9), Sf ( n = 10), and Sf:: Keap1 FA/− ( n = 10) mice. The horizontal bars represent the means. (B) Gene expression of Ifng in the LNs and spleens of WT ( n = 4), Keap1 FA/− ( n = 4), Sf ( n = 4), and Sf:: Keap1 FA/− ( n = 4) mice. The expression levels were normalized to Gapdh expression, and the expression level in WT mice was set to 1. The values represent the means and SD. NS, not significant; *, P

    Journal: Molecular and Cellular Biology

    Article Title: Systemic Activation of NRF2 Alleviates Lethal Autoimmune Inflammation in Scurfy Mice

    doi: 10.1128/MCB.00063-17

    Figure Lengend Snippet: Production of IFN-γ is dramatically reduced in Sf mice in the Keap1 knockdown background. (A) Serum cytokine levels in WT ( n = 10), Keap1 FA/− ( n = 9), Sf ( n = 10), and Sf:: Keap1 FA/− ( n = 10) mice. The horizontal bars represent the means. (B) Gene expression of Ifng in the LNs and spleens of WT ( n = 4), Keap1 FA/− ( n = 4), Sf ( n = 4), and Sf:: Keap1 FA/− ( n = 4) mice. The expression levels were normalized to Gapdh expression, and the expression level in WT mice was set to 1. The values represent the means and SD. NS, not significant; *, P

    Article Snippet: The samples were then subjected to immunoblot analysis using an anti-NRF2 antibody (clone 103; 1:500) , an anti-KEAP1 antibody (clone 111; 1:200) , and an anti-α-tubulin antibody (clone DM1A; 1:10,000) (Sigma-Aldrich).

    Techniques: Mouse Assay, Expressing

    T cell-specific Keap1 deletion decreases production of IFN-γ in T cells of Sf mice. (A) Frequencies of effector T cell subsets in the LNs of control ( n = 5), Sf::Control ( n = 8), and Sf:: Keap1 -TKO ( n = 5) mice. The percentages of the IFN-γ + , IL-4 + , and IL-17A + fractions of CD4 single-positive cells and the percentage of the IFN-γ + fraction of CD8 single-positive cells are shown. The values represent the means and SD. (B) Representative contour plot data for the IFN-γ + , IL-4 + , and IL-17A + fractions of CD4 single-positive LN cells and the IFN-γ + fraction of CD8 single-positive LN cells, with the percentages of cytokine-positive fractions shown. NS, not significant; *, P

    Journal: Molecular and Cellular Biology

    Article Title: Systemic Activation of NRF2 Alleviates Lethal Autoimmune Inflammation in Scurfy Mice

    doi: 10.1128/MCB.00063-17

    Figure Lengend Snippet: T cell-specific Keap1 deletion decreases production of IFN-γ in T cells of Sf mice. (A) Frequencies of effector T cell subsets in the LNs of control ( n = 5), Sf::Control ( n = 8), and Sf:: Keap1 -TKO ( n = 5) mice. The percentages of the IFN-γ + , IL-4 + , and IL-17A + fractions of CD4 single-positive cells and the percentage of the IFN-γ + fraction of CD8 single-positive cells are shown. The values represent the means and SD. (B) Representative contour plot data for the IFN-γ + , IL-4 + , and IL-17A + fractions of CD4 single-positive LN cells and the IFN-γ + fraction of CD8 single-positive LN cells, with the percentages of cytokine-positive fractions shown. NS, not significant; *, P

    Article Snippet: The samples were then subjected to immunoblot analysis using an anti-NRF2 antibody (clone 103; 1:500) , an anti-KEAP1 antibody (clone 111; 1:200) , and an anti-α-tubulin antibody (clone DM1A; 1:10,000) (Sigma-Aldrich).

    Techniques: Mouse Assay

    PHL targeted Keap1 to disassemble the Keap1/Nrf2 complex. (A) Per-residue of top 10 contribution to the binding free energy; (B) Structural analysis of the most 10 contributors of Keap1 to PHL, hydrogen bonds are colored yellow; (C) Alignment of the representative structures between from conventional MD simulation and from GaMD simulation; (D) PCA scatter plot of 200,000 snapshots from GaMD simulation along the first two principal components. (E) Immunoblotting analysis of Nrf2 expression and Co-Immunoprecipitation analysis of Nrf2 and Keap1 complex in lysates prepared from heart tissues. GAPDH was used as a loading control.

    Journal: Frontiers in Endocrinology

    Article Title: Phloretin Prevents Diabetic Cardiomyopathy by Dissociating Keap1/Nrf2 Complex and Inhibiting Oxidative Stress

    doi: 10.3389/fendo.2018.00774

    Figure Lengend Snippet: PHL targeted Keap1 to disassemble the Keap1/Nrf2 complex. (A) Per-residue of top 10 contribution to the binding free energy; (B) Structural analysis of the most 10 contributors of Keap1 to PHL, hydrogen bonds are colored yellow; (C) Alignment of the representative structures between from conventional MD simulation and from GaMD simulation; (D) PCA scatter plot of 200,000 snapshots from GaMD simulation along the first two principal components. (E) Immunoblotting analysis of Nrf2 expression and Co-Immunoprecipitation analysis of Nrf2 and Keap1 complex in lysates prepared from heart tissues. GAPDH was used as a loading control.

    Article Snippet: Antibodies for Nrf2 (#12721), TGF-β (#3711), Keap1 (#8047), GAPDH (#5174), and secondary antibodies (mouse #7076, rat #7077) were obtained from Cell Signaling Technology (Danvers, USA).

    Techniques: Binding Assay, Expressing, Immunoprecipitation

    Keap1, MCM3, and MCM-BP form a ternary complex. ( a ) Strep-Keap1 and FLAG-MCM3 pulldown experiments from Sf9 cells co-infected with baculoviruses expressing mouse MCM-BP together with WT or interaction deficient mutant MCM3 and Keap1 as indicated. Top panels show the Western blots of indicated proteins, bottom panel the blotted membranes that were stained with colloidal gold total protein stain. 1/300th of the starting extracts (‘input’) and 1/6th of the pulldown samples was loaded on each lane. See Supplementary Fig. S6 for full-length blots. ( b ) Strep-Keap1 - FLAG-MCM3 tandem affinity purification experiment from Sf9 cells co-infected with baculoviruses expressing all six mouse MCM2-7 subunits, Keap1, and MCM-BP. Coomassie brilliant blue stained SDS-PAGE gel on the left shows eluted material from both affinity purification steps, and unbound material from the FLAG affinity step in the middle lane. Resulting complexes were further resolved by Superose 6 size exclusion chromatography, the fractions of which are shown on right gel; co-elution of molecular weight markers is indicated at the bottom. The identity of protein bands was verified by mass spectrometry.

    Journal: Scientific Reports

    Article Title: Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa

    doi: 10.1038/s41598-018-30562-y

    Figure Lengend Snippet: Keap1, MCM3, and MCM-BP form a ternary complex. ( a ) Strep-Keap1 and FLAG-MCM3 pulldown experiments from Sf9 cells co-infected with baculoviruses expressing mouse MCM-BP together with WT or interaction deficient mutant MCM3 and Keap1 as indicated. Top panels show the Western blots of indicated proteins, bottom panel the blotted membranes that were stained with colloidal gold total protein stain. 1/300th of the starting extracts (‘input’) and 1/6th of the pulldown samples was loaded on each lane. See Supplementary Fig. S6 for full-length blots. ( b ) Strep-Keap1 - FLAG-MCM3 tandem affinity purification experiment from Sf9 cells co-infected with baculoviruses expressing all six mouse MCM2-7 subunits, Keap1, and MCM-BP. Coomassie brilliant blue stained SDS-PAGE gel on the left shows eluted material from both affinity purification steps, and unbound material from the FLAG affinity step in the middle lane. Resulting complexes were further resolved by Superose 6 size exclusion chromatography, the fractions of which are shown on right gel; co-elution of molecular weight markers is indicated at the bottom. The identity of protein bands was verified by mass spectrometry.

    Article Snippet: Antibodies used in Western blotting Antibodies against following proteins were used: MCM2 (N19, sc-9839), MCM3 (N19, sc-9850), MCM4 (C-10, sc-48407), MCM5 (33, sc-136366), MCM6 (H-300, sc-22780), MCM6 (C-20, sc-9843), MCM7 (H-5, sc-374403), Heme Oxygenase 1 (HO1) (A-3, sc-136960), actin (I-19, sc-1616), Histone H3 (FL-136, sc-10809), GAPDH (6C5, sc-32233), Lamin B1 (A-11, sc-377000), Sp1 (sc-59), and Keap1 (E-20, sc-15246) - all from Santa Cruz Biotechnology; Keap1 (D6B12), and Nrf2 (D1Z9C) - Cell Signaling Technology; MCM-BP (HPA038481), and anti-FLAG-tag (M2, F1804) - Sigma-Aldrich; anti- basal cell cytokeratin (RCK103, ab9222) - Abcam.

    Techniques: Infection, Expressing, Mutagenesis, Western Blot, Staining, Affinity Purification, SDS Page, Size-exclusion Chromatography, Co-Elution Assay, Molecular Weight, Mass Spectrometry

    MCM3 and Nrf2 bind to Keap1 in structurally highly similar and competitive manner. ( a ) Sequence alignment of the H2I beta hairpin motifs from human MCM2-7 and Sulfolobus solfataricus (Sso) MCM proteins. ( b ) A cartoon showing the conserved order of MCM subunits in MCM2-7 heterohexamer and H2I hairpins in the central channel. ( c ) Structure models of Saccharomyces cerevisiae single MCM2-7 complex on the left (PDB accession code 3JA8 38 ) and a Kelch domain of human Keap1 bound to DxETGE motif peptide from Nrf2 on the right (PDB accession code 2flu 22 ). Kelch domain (beige) is viewed from the side opposite to the binding pocket. MCM2-7 is shown as a top view on its N-terminal tier, MCM3 subunit coloured light blue and opposite MCM6 subunit green. The Keap1 interacting beta hairpin motifs of MCM3 and Nrf2 proteins are in dark blue and marked by boxes here and on panel ‘d’, with ETGE box residues presented by red sphere models. ( d ) Side view (horizontal clockwise 90° rotation) of the same models, where all the other MCM subunits apart from MCM3 and MCM6 have been removed to reveal the central channel of MCM2-7 ring. ( e ) Keap1 pulldown from baculovirus infected Sf9 cells co-expressing all six mouse MCM2-7 proteins and a strep tagged Keap1. Western blots show the protein levels in input extracts (left lanes) and in pulldown samples (right lanes) with co-expressed wt (‘+’) or interaction deficient mutant (‘mut’) proteins as indicated on top. Purified stoichiometric mouse MCM2-7 was loaded on the first lane (‘MCM2-7’) as a reference for comparing different MCM blots. 1/300th of the input extract and 1/6th of the pulldown samples were loaded on each lane. See Supplementary Fig. S4a for images of full-length blots. ( f ) Western blot analysis of Keap1 pulldown experiment from baculovirus co-infected Sf9 cells co-expressing Nrf2 and MCM3 proteins with strep tagged Keap1. Keap1-Nrf2-MCM3 viruses were co-infected at the ratio of 0.1: 0. 5: 3.0 See Supplementary Fig. S4b for images of full-length blots.

    Journal: Scientific Reports

    Article Title: Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa

    doi: 10.1038/s41598-018-30562-y

    Figure Lengend Snippet: MCM3 and Nrf2 bind to Keap1 in structurally highly similar and competitive manner. ( a ) Sequence alignment of the H2I beta hairpin motifs from human MCM2-7 and Sulfolobus solfataricus (Sso) MCM proteins. ( b ) A cartoon showing the conserved order of MCM subunits in MCM2-7 heterohexamer and H2I hairpins in the central channel. ( c ) Structure models of Saccharomyces cerevisiae single MCM2-7 complex on the left (PDB accession code 3JA8 38 ) and a Kelch domain of human Keap1 bound to DxETGE motif peptide from Nrf2 on the right (PDB accession code 2flu 22 ). Kelch domain (beige) is viewed from the side opposite to the binding pocket. MCM2-7 is shown as a top view on its N-terminal tier, MCM3 subunit coloured light blue and opposite MCM6 subunit green. The Keap1 interacting beta hairpin motifs of MCM3 and Nrf2 proteins are in dark blue and marked by boxes here and on panel ‘d’, with ETGE box residues presented by red sphere models. ( d ) Side view (horizontal clockwise 90° rotation) of the same models, where all the other MCM subunits apart from MCM3 and MCM6 have been removed to reveal the central channel of MCM2-7 ring. ( e ) Keap1 pulldown from baculovirus infected Sf9 cells co-expressing all six mouse MCM2-7 proteins and a strep tagged Keap1. Western blots show the protein levels in input extracts (left lanes) and in pulldown samples (right lanes) with co-expressed wt (‘+’) or interaction deficient mutant (‘mut’) proteins as indicated on top. Purified stoichiometric mouse MCM2-7 was loaded on the first lane (‘MCM2-7’) as a reference for comparing different MCM blots. 1/300th of the input extract and 1/6th of the pulldown samples were loaded on each lane. See Supplementary Fig. S4a for images of full-length blots. ( f ) Western blot analysis of Keap1 pulldown experiment from baculovirus co-infected Sf9 cells co-expressing Nrf2 and MCM3 proteins with strep tagged Keap1. Keap1-Nrf2-MCM3 viruses were co-infected at the ratio of 0.1: 0. 5: 3.0 See Supplementary Fig. S4b for images of full-length blots.

    Article Snippet: Antibodies used in Western blotting Antibodies against following proteins were used: MCM2 (N19, sc-9839), MCM3 (N19, sc-9850), MCM4 (C-10, sc-48407), MCM5 (33, sc-136366), MCM6 (H-300, sc-22780), MCM6 (C-20, sc-9843), MCM7 (H-5, sc-374403), Heme Oxygenase 1 (HO1) (A-3, sc-136960), actin (I-19, sc-1616), Histone H3 (FL-136, sc-10809), GAPDH (6C5, sc-32233), Lamin B1 (A-11, sc-377000), Sp1 (sc-59), and Keap1 (E-20, sc-15246) - all from Santa Cruz Biotechnology; Keap1 (D6B12), and Nrf2 (D1Z9C) - Cell Signaling Technology; MCM-BP (HPA038481), and anti-FLAG-tag (M2, F1804) - Sigma-Aldrich; anti- basal cell cytokeratin (RCK103, ab9222) - Abcam.

    Techniques: Sequencing, Binding Assay, Infection, Expressing, Western Blot, Mutagenesis, Purification

    siRNA knock-down of MCM3 levels results in lower sensitivity of Keap1 - Nrf2 response. ( a ) Western blotting analysis of human U2OS cells transfected with MCM3 siRNA #1, or negative control siRNA, and treated with indicated concentrations of tBHQ to induce the Keap1 controlled stabilization of Nrf2 protein. MCM3 blot shows the efficiency of a knock-down and actin blot serves as a loading control in all the panels of this figure. ( b ) Similar experiment, where different siRNA was used (#2) to knock down the MCM3 expression, and cells were treated with higher tBHQ concentrations. Nrf2 transactivation target heme oxygenase 1 (HO1) was additionally blotted. ( c ) The knock-down experiment with MCM3 siRNA #1, where different chemical activator (DEM) was used to induce the Keap1 controlled Nrf2 response. ( d ) Transfection experiments with U2OS cells showing the induction of Nrf2 levels in response to 50 µM DEM treatment (6 hrs) in cells over-expressing either WT or ETGE > GAGA mutant MCM3. Ectopically expressed MCM3 carried N-terminal FLAG and MBP tags and was blotted using antibodies against the FLAG tag of the protein.

    Journal: Scientific Reports

    Article Title: Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa

    doi: 10.1038/s41598-018-30562-y

    Figure Lengend Snippet: siRNA knock-down of MCM3 levels results in lower sensitivity of Keap1 - Nrf2 response. ( a ) Western blotting analysis of human U2OS cells transfected with MCM3 siRNA #1, or negative control siRNA, and treated with indicated concentrations of tBHQ to induce the Keap1 controlled stabilization of Nrf2 protein. MCM3 blot shows the efficiency of a knock-down and actin blot serves as a loading control in all the panels of this figure. ( b ) Similar experiment, where different siRNA was used (#2) to knock down the MCM3 expression, and cells were treated with higher tBHQ concentrations. Nrf2 transactivation target heme oxygenase 1 (HO1) was additionally blotted. ( c ) The knock-down experiment with MCM3 siRNA #1, where different chemical activator (DEM) was used to induce the Keap1 controlled Nrf2 response. ( d ) Transfection experiments with U2OS cells showing the induction of Nrf2 levels in response to 50 µM DEM treatment (6 hrs) in cells over-expressing either WT or ETGE > GAGA mutant MCM3. Ectopically expressed MCM3 carried N-terminal FLAG and MBP tags and was blotted using antibodies against the FLAG tag of the protein.

    Article Snippet: Antibodies used in Western blotting Antibodies against following proteins were used: MCM2 (N19, sc-9839), MCM3 (N19, sc-9850), MCM4 (C-10, sc-48407), MCM5 (33, sc-136366), MCM6 (H-300, sc-22780), MCM6 (C-20, sc-9843), MCM7 (H-5, sc-374403), Heme Oxygenase 1 (HO1) (A-3, sc-136960), actin (I-19, sc-1616), Histone H3 (FL-136, sc-10809), GAPDH (6C5, sc-32233), Lamin B1 (A-11, sc-377000), Sp1 (sc-59), and Keap1 (E-20, sc-15246) - all from Santa Cruz Biotechnology; Keap1 (D6B12), and Nrf2 (D1Z9C) - Cell Signaling Technology; MCM-BP (HPA038481), and anti-FLAG-tag (M2, F1804) - Sigma-Aldrich; anti- basal cell cytokeratin (RCK103, ab9222) - Abcam.

    Techniques: Western Blot, Transfection, Negative Control, Expressing, Mutagenesis, FLAG-tag

    Characterisation of Keap1-MCM3 interaction. ( a ) Strep-Keap1 and FLAG-MCM3 pulldown from the baculovirus infected cells expressing indicated combinations of mouse Keap1, MCM3, and MCM7 proteins. Western blots show the protein levels in input extracts (left lanes) and in pulldown samples (right lanes). WT (‘+’) or interaction deficient mutant (‘mut’) proteins were co-expressed as indicated on top. 1/300th of the input extract and 1/6th of the pulldown samples were loaded on each lane. See Supplementary Fig. S5 for images of full-length blots. ( b ) Coomassie brilliant blue stained SDS-PAGE gels of FLAG-MCM3 – strep-Keap1 tandem affinity pulldown (left panel), and strep-Keap1 – FLAG-MCM3 tandem affinity pull down (right panel) from the baculovirus infected Sf9 cells expressing mouse Keap1 and all six MCM2-7 subunit proteins. Lanes correspond to the eluted material from both pulldown steps and to the unbound material (‘flow’) from the second step as indicated.

    Journal: Scientific Reports

    Article Title: Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa

    doi: 10.1038/s41598-018-30562-y

    Figure Lengend Snippet: Characterisation of Keap1-MCM3 interaction. ( a ) Strep-Keap1 and FLAG-MCM3 pulldown from the baculovirus infected cells expressing indicated combinations of mouse Keap1, MCM3, and MCM7 proteins. Western blots show the protein levels in input extracts (left lanes) and in pulldown samples (right lanes). WT (‘+’) or interaction deficient mutant (‘mut’) proteins were co-expressed as indicated on top. 1/300th of the input extract and 1/6th of the pulldown samples were loaded on each lane. See Supplementary Fig. S5 for images of full-length blots. ( b ) Coomassie brilliant blue stained SDS-PAGE gels of FLAG-MCM3 – strep-Keap1 tandem affinity pulldown (left panel), and strep-Keap1 – FLAG-MCM3 tandem affinity pull down (right panel) from the baculovirus infected Sf9 cells expressing mouse Keap1 and all six MCM2-7 subunit proteins. Lanes correspond to the eluted material from both pulldown steps and to the unbound material (‘flow’) from the second step as indicated.

    Article Snippet: Antibodies used in Western blotting Antibodies against following proteins were used: MCM2 (N19, sc-9839), MCM3 (N19, sc-9850), MCM4 (C-10, sc-48407), MCM5 (33, sc-136366), MCM6 (H-300, sc-22780), MCM6 (C-20, sc-9843), MCM7 (H-5, sc-374403), Heme Oxygenase 1 (HO1) (A-3, sc-136960), actin (I-19, sc-1616), Histone H3 (FL-136, sc-10809), GAPDH (6C5, sc-32233), Lamin B1 (A-11, sc-377000), Sp1 (sc-59), and Keap1 (E-20, sc-15246) - all from Santa Cruz Biotechnology; Keap1 (D6B12), and Nrf2 (D1Z9C) - Cell Signaling Technology; MCM-BP (HPA038481), and anti-FLAG-tag (M2, F1804) - Sigma-Aldrich; anti- basal cell cytokeratin (RCK103, ab9222) - Abcam.

    Techniques: Infection, Expressing, Western Blot, Mutagenesis, Staining, SDS Page, Flow Cytometry

    Comparative evolutionary sequence analysis of the DxETGE interaction box in MCM3, Nrf2, and Nrf1 proteins. Sequence homology alignment of DxETGE interaction box and its beta hairpin context in the proteins from indicated species. Black vertical line between MCM3 and Nrf1 columns indicates the presence of Keap1 orthologue in the respective species.

    Journal: Scientific Reports

    Article Title: Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa

    doi: 10.1038/s41598-018-30562-y

    Figure Lengend Snippet: Comparative evolutionary sequence analysis of the DxETGE interaction box in MCM3, Nrf2, and Nrf1 proteins. Sequence homology alignment of DxETGE interaction box and its beta hairpin context in the proteins from indicated species. Black vertical line between MCM3 and Nrf1 columns indicates the presence of Keap1 orthologue in the respective species.

    Article Snippet: Antibodies used in Western blotting Antibodies against following proteins were used: MCM2 (N19, sc-9839), MCM3 (N19, sc-9850), MCM4 (C-10, sc-48407), MCM5 (33, sc-136366), MCM6 (H-300, sc-22780), MCM6 (C-20, sc-9843), MCM7 (H-5, sc-374403), Heme Oxygenase 1 (HO1) (A-3, sc-136960), actin (I-19, sc-1616), Histone H3 (FL-136, sc-10809), GAPDH (6C5, sc-32233), Lamin B1 (A-11, sc-377000), Sp1 (sc-59), and Keap1 (E-20, sc-15246) - all from Santa Cruz Biotechnology; Keap1 (D6B12), and Nrf2 (D1Z9C) - Cell Signaling Technology; MCM-BP (HPA038481), and anti-FLAG-tag (M2, F1804) - Sigma-Aldrich; anti- basal cell cytokeratin (RCK103, ab9222) - Abcam.

    Techniques: Sequencing

    Keap1 interacts with MCM3 in mammalian cells. ( a ) Western blots with antibodies against indicated proteins either with nuclear (‘N’) or cytoplasmic (‘C’) extracts of the FLAG-MCM3 expressing CHO-EBNALT85 cells (‘input’), or in MCM3 complexes immunoprecipitated with anti-FLAG affinity beads (‘flag IP’). Histone H3 and GAPDH were used as fractionation controls. See Supplementary Fig. S2a for full-length blots. ( b ) Coomassie brilliant blue stained SDS-PAGE gels (top panels) and Western blots with antibodies against indicated proteins (bottom panels) showing distribution of FLAG-MCM3 immunoprecipitated nuclear and cytoplasmic protein complexes in the Superdex 200 size exclusion chromatography. ‘flag’ depicts the lanes with input material. Co-elution of molecular weight markers is indicated at the bottom. See Supplementary Fig. S2b for full-length gels and blots. ( c ) Proximity ligation analysis (PLA) of the Keap1 - MCM3 interaction in human primary epithelial keratinocytes (HPEK). The images of red PLA channel alone are shown in the left column, and combined with blue DAPI staining of nuclei in the right column. ‘Keap1 + MCM3’ indicates the images with interaction specific signals, other images correspond to the control experiments with single antibodies. Shown are the maximum intensity projection images of the Z stacks from confocal microscopy; white scale bar = 10 µM. ( d ) Scatter dot plot of the quantified data of nuclear and cytoplasmic Keap1 + MCM3 PLA signals (M3 + K1) compared to negative control with MCM3 antibody alone (M3). Each data point represents an average number of nuclear or cytoplasmic PLA dots per cell from one micrograph. Bars represent the mean and standard deviation of combined data from two independent PLA experiments, one slide analysed in first and two in second experiment and three different micrographs quantified from each slide. The significance values (***p

    Journal: Scientific Reports

    Article Title: Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa

    doi: 10.1038/s41598-018-30562-y

    Figure Lengend Snippet: Keap1 interacts with MCM3 in mammalian cells. ( a ) Western blots with antibodies against indicated proteins either with nuclear (‘N’) or cytoplasmic (‘C’) extracts of the FLAG-MCM3 expressing CHO-EBNALT85 cells (‘input’), or in MCM3 complexes immunoprecipitated with anti-FLAG affinity beads (‘flag IP’). Histone H3 and GAPDH were used as fractionation controls. See Supplementary Fig. S2a for full-length blots. ( b ) Coomassie brilliant blue stained SDS-PAGE gels (top panels) and Western blots with antibodies against indicated proteins (bottom panels) showing distribution of FLAG-MCM3 immunoprecipitated nuclear and cytoplasmic protein complexes in the Superdex 200 size exclusion chromatography. ‘flag’ depicts the lanes with input material. Co-elution of molecular weight markers is indicated at the bottom. See Supplementary Fig. S2b for full-length gels and blots. ( c ) Proximity ligation analysis (PLA) of the Keap1 - MCM3 interaction in human primary epithelial keratinocytes (HPEK). The images of red PLA channel alone are shown in the left column, and combined with blue DAPI staining of nuclei in the right column. ‘Keap1 + MCM3’ indicates the images with interaction specific signals, other images correspond to the control experiments with single antibodies. Shown are the maximum intensity projection images of the Z stacks from confocal microscopy; white scale bar = 10 µM. ( d ) Scatter dot plot of the quantified data of nuclear and cytoplasmic Keap1 + MCM3 PLA signals (M3 + K1) compared to negative control with MCM3 antibody alone (M3). Each data point represents an average number of nuclear or cytoplasmic PLA dots per cell from one micrograph. Bars represent the mean and standard deviation of combined data from two independent PLA experiments, one slide analysed in first and two in second experiment and three different micrographs quantified from each slide. The significance values (***p

    Article Snippet: Antibodies used in Western blotting Antibodies against following proteins were used: MCM2 (N19, sc-9839), MCM3 (N19, sc-9850), MCM4 (C-10, sc-48407), MCM5 (33, sc-136366), MCM6 (H-300, sc-22780), MCM6 (C-20, sc-9843), MCM7 (H-5, sc-374403), Heme Oxygenase 1 (HO1) (A-3, sc-136960), actin (I-19, sc-1616), Histone H3 (FL-136, sc-10809), GAPDH (6C5, sc-32233), Lamin B1 (A-11, sc-377000), Sp1 (sc-59), and Keap1 (E-20, sc-15246) - all from Santa Cruz Biotechnology; Keap1 (D6B12), and Nrf2 (D1Z9C) - Cell Signaling Technology; MCM-BP (HPA038481), and anti-FLAG-tag (M2, F1804) - Sigma-Aldrich; anti- basal cell cytokeratin (RCK103, ab9222) - Abcam.

    Techniques: Western Blot, Expressing, Immunoprecipitation, Fractionation, Staining, SDS Page, Size-exclusion Chromatography, Co-Elution Assay, Molecular Weight, Ligation, Proximity Ligation Assay, Confocal Microscopy, Negative Control, Standard Deviation

    The presence of DxETGE or similar sequence box in the orthologues of characterized or known candidate interaction partners of human Keap1. Comparative evolutionary sequence analysis of the orthologues of identified and candidate partners of human Keap1 that contain ETGE or ESGE consensus motif, or similar DxSTGE motif in case of known Keap1 partner SQSTM1. The conservation is presented using following legend: dark green - ETGE in conserved position; medium green – T > S in human protein, or no more than two conservative E > D or T > S substitutions in other species; light green - one substitution of any other kind plus no more than one additional E > D or T > S substitution; ‘X’ indicates conserved D in -2 position. Grey boxes indicate orthologues with no or very little ETGE similarity, and black boxes in the first column the presence of a Keap1 orthologue. The species are indicated with KEGG organism codes and are listed in the same order as in Fig. 5 .

    Journal: Scientific Reports

    Article Title: Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa

    doi: 10.1038/s41598-018-30562-y

    Figure Lengend Snippet: The presence of DxETGE or similar sequence box in the orthologues of characterized or known candidate interaction partners of human Keap1. Comparative evolutionary sequence analysis of the orthologues of identified and candidate partners of human Keap1 that contain ETGE or ESGE consensus motif, or similar DxSTGE motif in case of known Keap1 partner SQSTM1. The conservation is presented using following legend: dark green - ETGE in conserved position; medium green – T > S in human protein, or no more than two conservative E > D or T > S substitutions in other species; light green - one substitution of any other kind plus no more than one additional E > D or T > S substitution; ‘X’ indicates conserved D in -2 position. Grey boxes indicate orthologues with no or very little ETGE similarity, and black boxes in the first column the presence of a Keap1 orthologue. The species are indicated with KEGG organism codes and are listed in the same order as in Fig. 5 .

    Article Snippet: Antibodies used in Western blotting Antibodies against following proteins were used: MCM2 (N19, sc-9839), MCM3 (N19, sc-9850), MCM4 (C-10, sc-48407), MCM5 (33, sc-136366), MCM6 (H-300, sc-22780), MCM6 (C-20, sc-9843), MCM7 (H-5, sc-374403), Heme Oxygenase 1 (HO1) (A-3, sc-136960), actin (I-19, sc-1616), Histone H3 (FL-136, sc-10809), GAPDH (6C5, sc-32233), Lamin B1 (A-11, sc-377000), Sp1 (sc-59), and Keap1 (E-20, sc-15246) - all from Santa Cruz Biotechnology; Keap1 (D6B12), and Nrf2 (D1Z9C) - Cell Signaling Technology; MCM-BP (HPA038481), and anti-FLAG-tag (M2, F1804) - Sigma-Aldrich; anti- basal cell cytokeratin (RCK103, ab9222) - Abcam.

    Techniques: Sequencing

    Keap1 inhibition improves ROS handling within fibroblasts by enhancing activation of the endogenous antioxidant program. Gene expression and ROS handling were assessed in vitro following Keap1 silencing. A : Chronic hyperglycemia impairs activation of NQO1 and MnSOD by 72% and 38%, respectively, compared with NG controls. B : siRNA-mediated reduction of Keap1 expression rescues MnSOD and NQO1 expression to 180% and 410% of controls, respectively. C–E : Gene expression analysis of Nrf2 target genes ( C ), growth factors ( D ), and inflammatory markers ( E ) with and without Keap1 silencing. F : Assessment of GSH/GSSG content with si Keap1 . G : CM-H 2 DCFDA–based fluorescent assessment of ROS production reveals that real-time ROS production in HG fibroblasts can be reduced to that of NG cells with si Keap1 . H : Keap1 inhibition within HG fibroblasts reduces the ROS by-product 8-OHdG by 62% compared with control. 8-OHdG in NG cells is nonsignificantly decreased by 25%. I : db/db Mice have 5.6-fold greater accumulation of ROS in wounded skin compared with WT controls; intact db/db skin accumulates 3.5-fold more ROS than wounded WT skin. * P

    Journal: Diabetes

    Article Title: Restoration of Nrf2 Signaling Normalizes the Regenerative Niche

    doi: 10.2337/db15-0453

    Figure Lengend Snippet: Keap1 inhibition improves ROS handling within fibroblasts by enhancing activation of the endogenous antioxidant program. Gene expression and ROS handling were assessed in vitro following Keap1 silencing. A : Chronic hyperglycemia impairs activation of NQO1 and MnSOD by 72% and 38%, respectively, compared with NG controls. B : siRNA-mediated reduction of Keap1 expression rescues MnSOD and NQO1 expression to 180% and 410% of controls, respectively. C–E : Gene expression analysis of Nrf2 target genes ( C ), growth factors ( D ), and inflammatory markers ( E ) with and without Keap1 silencing. F : Assessment of GSH/GSSG content with si Keap1 . G : CM-H 2 DCFDA–based fluorescent assessment of ROS production reveals that real-time ROS production in HG fibroblasts can be reduced to that of NG cells with si Keap1 . H : Keap1 inhibition within HG fibroblasts reduces the ROS by-product 8-OHdG by 62% compared with control. 8-OHdG in NG cells is nonsignificantly decreased by 25%. I : db/db Mice have 5.6-fold greater accumulation of ROS in wounded skin compared with WT controls; intact db/db skin accumulates 3.5-fold more ROS than wounded WT skin. * P

    Article Snippet: The tissue was probed using anti-Nrf2 antibody (sc-722; Santa Cruz Biotechnology) and anti-Keap1 antibody (sc-15246; Santa Cruz Biotechnology).

    Techniques: Inhibition, Activation Assay, Expressing, In Vitro, Mouse Assay

    Topical si Keap1 gene therapy improves the histologic profile of diabetic wounds. Ten-day-old diabetic wounds were analyzed histologically to study the effect of topical si Keap1 therapy on reepithelialization and neovascularization. A : Hematoxylin and eosin–stained sections demonstrate accelerated reepithelialization in si Keap1 -treated wounds. The yellow dotted lines indicate wound boundaries. B : Quantification of the epithelial gap. C : si Keap1 -treated diabetic wounds demonstrate increased granulation tissue area. The black arrows indicate granulation tissue. D : Quantification of granulation tissue. si Keap1 therapy increases granulation tissue production by > 3.5-fold compared with scramble-treated controls (* P

    Journal: Diabetes

    Article Title: Restoration of Nrf2 Signaling Normalizes the Regenerative Niche

    doi: 10.2337/db15-0453

    Figure Lengend Snippet: Topical si Keap1 gene therapy improves the histologic profile of diabetic wounds. Ten-day-old diabetic wounds were analyzed histologically to study the effect of topical si Keap1 therapy on reepithelialization and neovascularization. A : Hematoxylin and eosin–stained sections demonstrate accelerated reepithelialization in si Keap1 -treated wounds. The yellow dotted lines indicate wound boundaries. B : Quantification of the epithelial gap. C : si Keap1 -treated diabetic wounds demonstrate increased granulation tissue area. The black arrows indicate granulation tissue. D : Quantification of granulation tissue. si Keap1 therapy increases granulation tissue production by > 3.5-fold compared with scramble-treated controls (* P

    Article Snippet: The tissue was probed using anti-Nrf2 antibody (sc-722; Santa Cruz Biotechnology) and anti-Keap1 antibody (sc-15246; Santa Cruz Biotechnology).

    Techniques: Staining

    Defective Nrf2 nuclear translocation in chronic hyperglycemia can be alleviated by Keap1 inhibition. Protein lysates of 3T3 fibroblasts cultured in either NG or chronic HG conditions were generated 48 h after introduction of si Keap1 or control scramble siRNA. A : Cytoplasmic protein lysate (10 μg) from NG and chronic HG samples reveal relatively equivalent amounts of Nrf2 within the cytoplasm. B : Immunoprecipitation of cytosolic Keap1 demonstrates that there are notable differences between the proportions of Nrf2 sequestered by Keap1 in the cytoplasm between NG and HG 3T3s. C : Quantitative RT-PCR demonstrates that Lipofectamine-based si Keap1 transfection effectively reduces Keap1 expression in cultured fibroblasts to

    Journal: Diabetes

    Article Title: Restoration of Nrf2 Signaling Normalizes the Regenerative Niche

    doi: 10.2337/db15-0453

    Figure Lengend Snippet: Defective Nrf2 nuclear translocation in chronic hyperglycemia can be alleviated by Keap1 inhibition. Protein lysates of 3T3 fibroblasts cultured in either NG or chronic HG conditions were generated 48 h after introduction of si Keap1 or control scramble siRNA. A : Cytoplasmic protein lysate (10 μg) from NG and chronic HG samples reveal relatively equivalent amounts of Nrf2 within the cytoplasm. B : Immunoprecipitation of cytosolic Keap1 demonstrates that there are notable differences between the proportions of Nrf2 sequestered by Keap1 in the cytoplasm between NG and HG 3T3s. C : Quantitative RT-PCR demonstrates that Lipofectamine-based si Keap1 transfection effectively reduces Keap1 expression in cultured fibroblasts to

    Article Snippet: The tissue was probed using anti-Nrf2 antibody (sc-722; Santa Cruz Biotechnology) and anti-Keap1 antibody (sc-15246; Santa Cruz Biotechnology).

    Techniques: Translocation Assay, Inhibition, Cell Culture, Generated, Immunoprecipitation, Quantitative RT-PCR, Transfection, Expressing

    Keap1 silencing in vivo upregulates Nrf2-mediated antioxidant mechanisms. Wound healing in db/db mice was analyzed using a validated stented-wound model, and the effect of weekly topical si Keap1 therapy was evaluated on macroscopic and molecular levels. A : Schematic of topical si Keap1 therapy. B : mRNA from si Keap1 -treated wounds shows a 36% reduction in Keap1 expression 10 days into treatment. C : Ten days into treatment, topical si Keap1 therapy increases NQO1 expression in wounds by 73% compared with scramble siRNA. D : MnSOD expression in 10-day treated diabetic wounds. E–G : Gene expression of Nrf2 target genes ( E ), growth factors ( F ), and inflammatory factors ( G ) in wound beds following si Keap1 topical therapy. H–M : Immunofluorescence of Nrf2 and Keap1 in tissue sections of wounded diabetic tissue, with indicated topical siRNA therapy. All images are ×10 magnification. Insets in J and M are ×20 magnification. Open arrowheads show low expression in the indicated region; white arrows show upregulated expression in the indicated region; asterisks note autofluorescence. The dotted line demarcates the epidermis (above) and dermis (below) at the wound edge. PDGF, platelet-derived growth factor; siNS, nonsense siRNA. * P

    Journal: Diabetes

    Article Title: Restoration of Nrf2 Signaling Normalizes the Regenerative Niche

    doi: 10.2337/db15-0453

    Figure Lengend Snippet: Keap1 silencing in vivo upregulates Nrf2-mediated antioxidant mechanisms. Wound healing in db/db mice was analyzed using a validated stented-wound model, and the effect of weekly topical si Keap1 therapy was evaluated on macroscopic and molecular levels. A : Schematic of topical si Keap1 therapy. B : mRNA from si Keap1 -treated wounds shows a 36% reduction in Keap1 expression 10 days into treatment. C : Ten days into treatment, topical si Keap1 therapy increases NQO1 expression in wounds by 73% compared with scramble siRNA. D : MnSOD expression in 10-day treated diabetic wounds. E–G : Gene expression of Nrf2 target genes ( E ), growth factors ( F ), and inflammatory factors ( G ) in wound beds following si Keap1 topical therapy. H–M : Immunofluorescence of Nrf2 and Keap1 in tissue sections of wounded diabetic tissue, with indicated topical siRNA therapy. All images are ×10 magnification. Insets in J and M are ×20 magnification. Open arrowheads show low expression in the indicated region; white arrows show upregulated expression in the indicated region; asterisks note autofluorescence. The dotted line demarcates the epidermis (above) and dermis (below) at the wound edge. PDGF, platelet-derived growth factor; siNS, nonsense siRNA. * P

    Article Snippet: The tissue was probed using anti-Nrf2 antibody (sc-722; Santa Cruz Biotechnology) and anti-Keap1 antibody (sc-15246; Santa Cruz Biotechnology).

    Techniques: In Vivo, Mouse Assay, Expressing, Immunofluorescence, Derivative Assay

    Topical si Keap1 gene therapy accelerates diabetic wound closure. A : Photographs of stented wounds demonstrating accelerated wound closure with weekly topical si Keap1 therapy. B : Topical si Keap1 therapy accelerates diabetic wound closure by 7 days compared with nontreated controls. C : Wound burden analysis demonstrates that topical si Keap1 therapy enhances diabetic wound healing by 49% compared with scramble control. D : Dermal penetration and accumulation of siRNA in cryosections of diabetic mouse skin treated with siGLO Red–liposomal complex. At 10 days after application, siGLO Red is present up to the panniculus carnosus. Scale bar, 100 μm. Inset is magnification of area in dashed box. E : In vivo imaging system imaging of 7-day-old wounds using systemically delivered L-012 reagent demonstrates that topical si Keap1 treatment reduces real-time ROS accumulation compared with control diabetic wounds. F : Quantification of L-012 bioluminescence demonstrating a 58% decrease in ROS levels within diabetic wounds treated with topical si Keap1 therapy. G : The downstream ROS by-product 8-OHdG is also reduced by 42% with topical si Keap1 therapy compared with scramble-treated wounds. d, day. * P

    Journal: Diabetes

    Article Title: Restoration of Nrf2 Signaling Normalizes the Regenerative Niche

    doi: 10.2337/db15-0453

    Figure Lengend Snippet: Topical si Keap1 gene therapy accelerates diabetic wound closure. A : Photographs of stented wounds demonstrating accelerated wound closure with weekly topical si Keap1 therapy. B : Topical si Keap1 therapy accelerates diabetic wound closure by 7 days compared with nontreated controls. C : Wound burden analysis demonstrates that topical si Keap1 therapy enhances diabetic wound healing by 49% compared with scramble control. D : Dermal penetration and accumulation of siRNA in cryosections of diabetic mouse skin treated with siGLO Red–liposomal complex. At 10 days after application, siGLO Red is present up to the panniculus carnosus. Scale bar, 100 μm. Inset is magnification of area in dashed box. E : In vivo imaging system imaging of 7-day-old wounds using systemically delivered L-012 reagent demonstrates that topical si Keap1 treatment reduces real-time ROS accumulation compared with control diabetic wounds. F : Quantification of L-012 bioluminescence demonstrating a 58% decrease in ROS levels within diabetic wounds treated with topical si Keap1 therapy. G : The downstream ROS by-product 8-OHdG is also reduced by 42% with topical si Keap1 therapy compared with scramble-treated wounds. d, day. * P

    Article Snippet: The tissue was probed using anti-Nrf2 antibody (sc-722; Santa Cruz Biotechnology) and anti-Keap1 antibody (sc-15246; Santa Cruz Biotechnology).

    Techniques: In Vivo Imaging, Imaging

    Keap1/Nrf2/HO-1 pathway inhibition alleviates the protective effects of rHMGB1 preconditioning in LIRI. a Western blot of nuclear Keap1 in lung tissues. b Western blot of nuclear Nrf2 in lung tissues. c Western blot of cytosolic HO-1 in lung tissues. d Morphological changes across groups observed using H E staining. Magnification, ×200. e Lung injury scoring. f Wet/dry ratios in lung tissues. g IL-1β abundance in lung tissues. h IL-6 abundance in lung tissues. i NF-κB abundance in lung tissues. (*p

    Journal: Journal of Translational Medicine

    Article Title: Preconditioning with rHMGB1 ameliorates lung ischemia–reperfusion injury by inhibiting alveolar macrophage pyroptosis via the Keap1/Nrf2/HO-1 signaling pathway

    doi: 10.1186/s12967-020-02467-w

    Figure Lengend Snippet: Keap1/Nrf2/HO-1 pathway inhibition alleviates the protective effects of rHMGB1 preconditioning in LIRI. a Western blot of nuclear Keap1 in lung tissues. b Western blot of nuclear Nrf2 in lung tissues. c Western blot of cytosolic HO-1 in lung tissues. d Morphological changes across groups observed using H E staining. Magnification, ×200. e Lung injury scoring. f Wet/dry ratios in lung tissues. g IL-1β abundance in lung tissues. h IL-6 abundance in lung tissues. i NF-κB abundance in lung tissues. (*p

    Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against Keap1 (1:200; Santa Cruz, CA, USA), Nrf2 (1:200; Santa Cruz), HO-1 (1:200; Santa Cruz), HMGB1 (1:1000; rabbit polyclonal, Abcam), β-actin (1:5000; mouse monoclonal, Abcam), and lamin A (1:1000; rabbit polyclonal, Abcam).

    Techniques: Inhibition, Western Blot, Staining

    Keap1/Nrf2/HO-1 pathway inhibition can suppress the anti-oxidant effects of rHMGB1 preconditioning in LIRI. a ROS. b MDA. c 15-F2t-isoprostane. d SOD. e GSH-PX. f CAT. (*p

    Journal: Journal of Translational Medicine

    Article Title: Preconditioning with rHMGB1 ameliorates lung ischemia–reperfusion injury by inhibiting alveolar macrophage pyroptosis via the Keap1/Nrf2/HO-1 signaling pathway

    doi: 10.1186/s12967-020-02467-w

    Figure Lengend Snippet: Keap1/Nrf2/HO-1 pathway inhibition can suppress the anti-oxidant effects of rHMGB1 preconditioning in LIRI. a ROS. b MDA. c 15-F2t-isoprostane. d SOD. e GSH-PX. f CAT. (*p

    Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against Keap1 (1:200; Santa Cruz, CA, USA), Nrf2 (1:200; Santa Cruz), HO-1 (1:200; Santa Cruz), HMGB1 (1:1000; rabbit polyclonal, Abcam), β-actin (1:5000; mouse monoclonal, Abcam), and lamin A (1:1000; rabbit polyclonal, Abcam).

    Techniques: Inhibition, Multiple Displacement Amplification

    rHMGB1 preconditioning inhibits AM pyroptosis via the Keap1/Nrf2/HO-1 pathway in LIRI. a Isolated AM counts in BALF. b LDH release from isolated AMs in BALF. c Representative results of flow cytometry assessing macrophage pyroptosis: F4/80 + cells were gated and analyzed for fluorescently labeled active caspase (FLICA) and propidium iodide (PI). d Quantitative analysis of F4/80 + FLICA + PI + cells. e Representative immunolabelling images for GSDMD protein from isolated AMs in BALF. f GSDMD levels in isolated AMs. (*p

    Journal: Journal of Translational Medicine

    Article Title: Preconditioning with rHMGB1 ameliorates lung ischemia–reperfusion injury by inhibiting alveolar macrophage pyroptosis via the Keap1/Nrf2/HO-1 signaling pathway

    doi: 10.1186/s12967-020-02467-w

    Figure Lengend Snippet: rHMGB1 preconditioning inhibits AM pyroptosis via the Keap1/Nrf2/HO-1 pathway in LIRI. a Isolated AM counts in BALF. b LDH release from isolated AMs in BALF. c Representative results of flow cytometry assessing macrophage pyroptosis: F4/80 + cells were gated and analyzed for fluorescently labeled active caspase (FLICA) and propidium iodide (PI). d Quantitative analysis of F4/80 + FLICA + PI + cells. e Representative immunolabelling images for GSDMD protein from isolated AMs in BALF. f GSDMD levels in isolated AMs. (*p

    Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against Keap1 (1:200; Santa Cruz, CA, USA), Nrf2 (1:200; Santa Cruz), HO-1 (1:200; Santa Cruz), HMGB1 (1:1000; rabbit polyclonal, Abcam), β-actin (1:5000; mouse monoclonal, Abcam), and lamin A (1:1000; rabbit polyclonal, Abcam).

    Techniques: Isolation, Affinity Magnetic Separation, Flow Cytometry, Labeling

    rHMGB1 preconditioning mediates the activity of the Keap1/Nrf2/HO-1 pathway in a mouse model of lung I/R. a Representative western blot images of nuclear Keap1 in lung tissues. b Nuclear Keap1 levels in lung tissues. c Representative western blot images of nuclear Nrf2 in lung tissues. d Nuclear Nrf2 expression levels in lung tissues. e Representative western blot images of cytosolic HO-1 in lung tissues. f Cytosolic HO-1 expression levels in lung tissues. (*p

    Journal: Journal of Translational Medicine

    Article Title: Preconditioning with rHMGB1 ameliorates lung ischemia–reperfusion injury by inhibiting alveolar macrophage pyroptosis via the Keap1/Nrf2/HO-1 signaling pathway

    doi: 10.1186/s12967-020-02467-w

    Figure Lengend Snippet: rHMGB1 preconditioning mediates the activity of the Keap1/Nrf2/HO-1 pathway in a mouse model of lung I/R. a Representative western blot images of nuclear Keap1 in lung tissues. b Nuclear Keap1 levels in lung tissues. c Representative western blot images of nuclear Nrf2 in lung tissues. d Nuclear Nrf2 expression levels in lung tissues. e Representative western blot images of cytosolic HO-1 in lung tissues. f Cytosolic HO-1 expression levels in lung tissues. (*p

    Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against Keap1 (1:200; Santa Cruz, CA, USA), Nrf2 (1:200; Santa Cruz), HO-1 (1:200; Santa Cruz), HMGB1 (1:1000; rabbit polyclonal, Abcam), β-actin (1:5000; mouse monoclonal, Abcam), and lamin A (1:1000; rabbit polyclonal, Abcam).

    Techniques: Activity Assay, Western Blot, Expressing

    miR-29 regulates Keap1 expression in HK-2 cells. a Cells were transfected with miR-29 mimic and b miR-29 inhibitor as well as respective negative control (NC) for 48 h; miR-29 level, translational activity of Keap1 and expression of Keap1 mRNA and protein were determined. Data were presented as mean ± S.D. ** P

    Journal: Journal of Translational Medicine

    Article Title: High glucose induces renal tubular epithelial injury via Sirt1/NF-kappaB/microR-29/Keap1 signal pathway

    doi: 10.1186/s12967-015-0710-y

    Figure Lengend Snippet: miR-29 regulates Keap1 expression in HK-2 cells. a Cells were transfected with miR-29 mimic and b miR-29 inhibitor as well as respective negative control (NC) for 48 h; miR-29 level, translational activity of Keap1 and expression of Keap1 mRNA and protein were determined. Data were presented as mean ± S.D. ** P

    Article Snippet: Nonspecific protein was blocked by incubation with 5 % nonfat dry milk in TBS for 2 h. Then membrane was blotted by rabbit polyclonal antibodies against acetyl-lysine p65 (1:1000; Thermo Fisher Scientific, USA), Keap1 (1:1000; Abcam, USA), GST (1:500; Santa Cruz biotechnology, CA, USA), NQO1 (1:500; Santa Cruz biotechnology, CA, USA) and Nrf2 (1:1000; Abcam, USA) for 16 h at 4 °C. β-actin was used as loading control for cytoplasmic proteins and α-tubulin was used as loading control for nuclear protein.

    Techniques: Expressing, Transfection, Negative Control, Activity Assay

    Ubiquitination of Nrf2 was regulated by miR-29/Keap1 axis in high glucose triggered HK-2 cells. a HK-2 miR−29mimic cells were transfected with Flag-ubiquitin and myc-Keap1 for 2 h and exposed to 5.5 and 45 mM glucose for 48 h; expression of ubiquitinated Keap1 protein was determined using western blot. b Western blot was performed to analyze expression of Keap1 and nuclear Nrf2 in 5.5 mM and 45 mM glucose-triggered HK-2 miR−29mimic cells

    Journal: Journal of Translational Medicine

    Article Title: High glucose induces renal tubular epithelial injury via Sirt1/NF-kappaB/microR-29/Keap1 signal pathway

    doi: 10.1186/s12967-015-0710-y

    Figure Lengend Snippet: Ubiquitination of Nrf2 was regulated by miR-29/Keap1 axis in high glucose triggered HK-2 cells. a HK-2 miR−29mimic cells were transfected with Flag-ubiquitin and myc-Keap1 for 2 h and exposed to 5.5 and 45 mM glucose for 48 h; expression of ubiquitinated Keap1 protein was determined using western blot. b Western blot was performed to analyze expression of Keap1 and nuclear Nrf2 in 5.5 mM and 45 mM glucose-triggered HK-2 miR−29mimic cells

    Article Snippet: Nonspecific protein was blocked by incubation with 5 % nonfat dry milk in TBS for 2 h. Then membrane was blotted by rabbit polyclonal antibodies against acetyl-lysine p65 (1:1000; Thermo Fisher Scientific, USA), Keap1 (1:1000; Abcam, USA), GST (1:500; Santa Cruz biotechnology, CA, USA), NQO1 (1:500; Santa Cruz biotechnology, CA, USA) and Nrf2 (1:1000; Abcam, USA) for 16 h at 4 °C. β-actin was used as loading control for cytoplasmic proteins and α-tubulin was used as loading control for nuclear protein.

    Techniques: Transfection, Expressing, Western Blot

    Correlation between blood parameters and signal molecules in diabetic rats. The rat model of diabetes was established by intraperitoneal injection of 65 mg/kg STZ. On week 0, 4, 8, 12, 16, a Serum creatinine and b blood urea nitrogen (BUN) were measured, and c renal tubule miR-29 level was determined using quantitative RT-PCR. Correlated analysis was performed to assess relationship between d miR-29 level and Serum creatinine as well as e miR-29 level and creatinine clearance. f Sirt1 activity was assessed. g Western blotting was performed to determine expressions of Keap1 and nuclear Nrf2 in isolated renal tubules. Data were presented as mean ± SD. **P

    Journal: Journal of Translational Medicine

    Article Title: High glucose induces renal tubular epithelial injury via Sirt1/NF-kappaB/microR-29/Keap1 signal pathway

    doi: 10.1186/s12967-015-0710-y

    Figure Lengend Snippet: Correlation between blood parameters and signal molecules in diabetic rats. The rat model of diabetes was established by intraperitoneal injection of 65 mg/kg STZ. On week 0, 4, 8, 12, 16, a Serum creatinine and b blood urea nitrogen (BUN) were measured, and c renal tubule miR-29 level was determined using quantitative RT-PCR. Correlated analysis was performed to assess relationship between d miR-29 level and Serum creatinine as well as e miR-29 level and creatinine clearance. f Sirt1 activity was assessed. g Western blotting was performed to determine expressions of Keap1 and nuclear Nrf2 in isolated renal tubules. Data were presented as mean ± SD. **P

    Article Snippet: Nonspecific protein was blocked by incubation with 5 % nonfat dry milk in TBS for 2 h. Then membrane was blotted by rabbit polyclonal antibodies against acetyl-lysine p65 (1:1000; Thermo Fisher Scientific, USA), Keap1 (1:1000; Abcam, USA), GST (1:500; Santa Cruz biotechnology, CA, USA), NQO1 (1:500; Santa Cruz biotechnology, CA, USA) and Nrf2 (1:1000; Abcam, USA) for 16 h at 4 °C. β-actin was used as loading control for cytoplasmic proteins and α-tubulin was used as loading control for nuclear protein.

    Techniques: Injection, Quantitative RT-PCR, Activity Assay, Western Blot, Isolation

    Effect of high glucose on renal tubule epithelia cell of HK-2 in vitro. Cells were triggered with doses of glucose (5.5, 15, 30 and 45) for 48 h. a Sirt1 activity was assessed. b NF-κB transcription activity was evaluated using luciferase reporter gene assay. c miR-29 expression was determined. d Western blot was performed to assess Keap1, GST, NQO1 and nuclear Nrf-2 expression. e Cell viability was evaluated using MTT assay. Data were presented as mean ± S.D. **P

    Journal: Journal of Translational Medicine

    Article Title: High glucose induces renal tubular epithelial injury via Sirt1/NF-kappaB/microR-29/Keap1 signal pathway

    doi: 10.1186/s12967-015-0710-y

    Figure Lengend Snippet: Effect of high glucose on renal tubule epithelia cell of HK-2 in vitro. Cells were triggered with doses of glucose (5.5, 15, 30 and 45) for 48 h. a Sirt1 activity was assessed. b NF-κB transcription activity was evaluated using luciferase reporter gene assay. c miR-29 expression was determined. d Western blot was performed to assess Keap1, GST, NQO1 and nuclear Nrf-2 expression. e Cell viability was evaluated using MTT assay. Data were presented as mean ± S.D. **P

    Article Snippet: Nonspecific protein was blocked by incubation with 5 % nonfat dry milk in TBS for 2 h. Then membrane was blotted by rabbit polyclonal antibodies against acetyl-lysine p65 (1:1000; Thermo Fisher Scientific, USA), Keap1 (1:1000; Abcam, USA), GST (1:500; Santa Cruz biotechnology, CA, USA), NQO1 (1:500; Santa Cruz biotechnology, CA, USA) and Nrf2 (1:1000; Abcam, USA) for 16 h at 4 °C. β-actin was used as loading control for cytoplasmic proteins and α-tubulin was used as loading control for nuclear protein.

    Techniques: In Vitro, Activity Assay, Luciferase, Reporter Gene Assay, Expressing, Western Blot, MTT Assay

    Expression of Keap1 Protein in the 6 experimental groups of mice. The upper bands (a) depict representative findings in the control, DIO, DEHP, DIO + DEHP, DIO + DEHP middle, and DIO + DEHP high groups. The lower bar graphs (b) show the results of the semiquantitative measurement of Keap1. Each bar represents mean ± SE. n = 4. ∗ indicates a significant difference from the control group, P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Di-(2-Ethylhexyl) Phthalate Increases Obesity-Induced Damage to the Male Reproductive System in Mice

    doi: 10.1155/2018/1861984

    Figure Lengend Snippet: Expression of Keap1 Protein in the 6 experimental groups of mice. The upper bands (a) depict representative findings in the control, DIO, DEHP, DIO + DEHP, DIO + DEHP middle, and DIO + DEHP high groups. The lower bar graphs (b) show the results of the semiquantitative measurement of Keap1. Each bar represents mean ± SE. n = 4. ∗ indicates a significant difference from the control group, P

    Article Snippet: After blocking in PBST containing 4% skimmed milk for 2 h at room temperature, the polyvinylidene fluoride membranes were incubated with rabbit polyclonal anti-Nrf2 antibody (ab31163, diluted 1 : 1000; Abcam) and anti-keap1 antibodies (ab119403, diluted 1 : 1000; Abcam) in PBST overnight at 4°C.

    Techniques: Expressing, Mouse Assay

    MsrA deletion abrogates the interaction of Nrf2 with Keap1, resulting in Nrf2 nuclear localization and transcriptional activity . (A, B) Representative immunoblots (A) and quantitation (B) of the interaction of Keap1 with p62 in MsrA-/- and WT VSMC. Immunoprecipitation for Keap1, immunoblots for p62 and Keap1. n = 6 biological replicates. (C, D) Representative immunoblots (C) and quantitation (D) of the interaction of Keap1 with Nrf2. Immunoprecipitation for Nrf2, immunoblots for Keap1 and Nrf2. n = 4 biological replicates. (E-G) Representative immunoblot (E) and summary data for Nrf2 localization in nuclear (F) and cytoplasmic fractions (G) of MsrA-/- and WT VSMC. TOPO IIβ: nuclear marker; GAPDH: cytoplasmic marker. n = 7 biological replicates. (H) Nrf2-dependent transcriptional activity as determined by quantification of ARE-dependent luciferase activity in MsrA-/- and WT VSMC expressing ARE-luciferase reporter. n = 6 biological replicates. * p

    Journal: Redox Biology

    Article Title: Defective protein repair under methionine sulfoxide A deletion drives autophagy and ARE-dependent gene transcription

    doi: 10.1016/j.redox.2018.04.001

    Figure Lengend Snippet: MsrA deletion abrogates the interaction of Nrf2 with Keap1, resulting in Nrf2 nuclear localization and transcriptional activity . (A, B) Representative immunoblots (A) and quantitation (B) of the interaction of Keap1 with p62 in MsrA-/- and WT VSMC. Immunoprecipitation for Keap1, immunoblots for p62 and Keap1. n = 6 biological replicates. (C, D) Representative immunoblots (C) and quantitation (D) of the interaction of Keap1 with Nrf2. Immunoprecipitation for Nrf2, immunoblots for Keap1 and Nrf2. n = 4 biological replicates. (E-G) Representative immunoblot (E) and summary data for Nrf2 localization in nuclear (F) and cytoplasmic fractions (G) of MsrA-/- and WT VSMC. TOPO IIβ: nuclear marker; GAPDH: cytoplasmic marker. n = 7 biological replicates. (H) Nrf2-dependent transcriptional activity as determined by quantification of ARE-dependent luciferase activity in MsrA-/- and WT VSMC expressing ARE-luciferase reporter. n = 6 biological replicates. * p

    Article Snippet: The following primary antibodies were used in this study: anti-dinitrophenol (#S7150, as part of the OxyBlot Protein Oxidation Detection Kit, EMD Millipore, 1:150), anti-SQSTM1/p62 (#7695, Cell Signaling, 1:500), anti-Nrf2 (#D1Z9C, Cell Signaling, 1:1000), anti-Keap1 (#ab150654, Abcam, 1:1000), anti-TOPO II β (#ab125297, Abcam, 1:1000), anti-LC3 (#D50G8, Cell Signaling, 1:1000), anti-ubiquitin (#sc-8017, Santa Cruz, 1:250), anti-GCLC (#PA5–44189, Invitrogen, 1:1000), anti-glutathione reductase (#ab16801, Abcam, 1:1000), anti-Bcl2 (#sc-7382 Santa Cruz, 1:1000), anti-GAPDH (#5174, Cell Signaling, 1:2,000), and anti-actin (#sc-1616, Santa Cruz, 1:500).

    Techniques: Activity Assay, Western Blot, Quantitation Assay, Immunoprecipitation, Marker, Luciferase, Expressing

    Chrysin deactivates Nrf2 signaling pathway in a Keap1-independent manner. ( A and B ) The relative protein levels of chrysin-treated cells were expressed compared with the vehicle-treated group. ( C ) Cells were processed with shRNA (Sc) or Nrf2 shRNA (Nrf2i). Reduced expression of Nrf2 was observed after exposure to Nrf2 shRNA. ( E and F ) Chrysin was unable to change protein levels of Nrf2 and Nrf2-target genes in U87 cells with Nrf2 knockdown. The cells were pretreated with Nrf2 shRNA (Nrf2i), followed by chrysin treatment for 24 hours. ( D ) Nrf2 knockdown decreased the sensitivity of cells to chrysin. Relative cell numbers were monitored by a CCK-8 assay. The seeded cells were adjusted to the value of 1. Data are expressed as mean ± SD (n=4). * p

    Journal: Drug Design, Development and Therapy

    Article Title: Chrysin suppresses proliferation, migration, and invasion in glioblastoma cell lines via mediating the ERK/Nrf2 signaling pathway

    doi: 10.2147/DDDT.S160020

    Figure Lengend Snippet: Chrysin deactivates Nrf2 signaling pathway in a Keap1-independent manner. ( A and B ) The relative protein levels of chrysin-treated cells were expressed compared with the vehicle-treated group. ( C ) Cells were processed with shRNA (Sc) or Nrf2 shRNA (Nrf2i). Reduced expression of Nrf2 was observed after exposure to Nrf2 shRNA. ( E and F ) Chrysin was unable to change protein levels of Nrf2 and Nrf2-target genes in U87 cells with Nrf2 knockdown. The cells were pretreated with Nrf2 shRNA (Nrf2i), followed by chrysin treatment for 24 hours. ( D ) Nrf2 knockdown decreased the sensitivity of cells to chrysin. Relative cell numbers were monitored by a CCK-8 assay. The seeded cells were adjusted to the value of 1. Data are expressed as mean ± SD (n=4). * p

    Article Snippet: Nrf2, HO-1, NQO-1, and Keap1 antibody were from Abcam (Cambridge, MA, USA).

    Techniques: shRNA, Expressing, CCK-8 Assay

    Keap1 inhibition and protein degradation pathways in Aβ42 flies. (A) Keap1 did not modify autophagy in Aβ42-expressing flies, as measured by the ratio of ATG8-II to ATG8-I levels. Data are presented as means ± SEM. P > 0.05 (one-way ANOVA and Tukey’s post-hoc analysis). N = 4 biological repeats of 10 fly heads per condition. (B) Proteasome activity, as measured using the fluorogenic peptide substrate LLVY-AMC, was increased by Keap1 modification in Aβ42-expressing flies. Data are presented as mean activities (pmoles/min/mg protein) ± SEM. P

    Journal: PLoS Genetics

    Article Title: Direct Keap1-Nrf2 disruption as a potential therapeutic target for Alzheimer’s disease

    doi: 10.1371/journal.pgen.1006593

    Figure Lengend Snippet: Keap1 inhibition and protein degradation pathways in Aβ42 flies. (A) Keap1 did not modify autophagy in Aβ42-expressing flies, as measured by the ratio of ATG8-II to ATG8-I levels. Data are presented as means ± SEM. P > 0.05 (one-way ANOVA and Tukey’s post-hoc analysis). N = 4 biological repeats of 10 fly heads per condition. (B) Proteasome activity, as measured using the fluorogenic peptide substrate LLVY-AMC, was increased by Keap1 modification in Aβ42-expressing flies. Data are presented as mean activities (pmoles/min/mg protein) ± SEM. P

    Article Snippet: Plates were fixed with 4% PFA (v/v) for 15 minutes, washed three times with PBS, permeabilized with 0.5% Triton-X before staining with primary antibodies against Keap1 (1:50; ab150654, Abcam) and Nrf2 (1:100; ab62352, Abcam) in blocking solution containing 10% goat serum and 3% BSA overnight at 4 C. After 3 washes in PBS secondary antibodies (α rabbit Alexa 594, ab150080, α mouse FITC, ab6785, Abcam) were applied.

    Techniques: Inhibition, Expressing, Activity Assay, Modification

    Effects of Keap1 inhibition or lithium treatment on sensitivity of Aβ42 flies to oxidative and xenobiotic stress. (A) Keap1 deletion and lithium treatment combined to protect against sensitivity to xenobiotic damage in Aβ42-expressing flies ( p

    Journal: PLoS Genetics

    Article Title: Direct Keap1-Nrf2 disruption as a potential therapeutic target for Alzheimer’s disease

    doi: 10.1371/journal.pgen.1006593

    Figure Lengend Snippet: Effects of Keap1 inhibition or lithium treatment on sensitivity of Aβ42 flies to oxidative and xenobiotic stress. (A) Keap1 deletion and lithium treatment combined to protect against sensitivity to xenobiotic damage in Aβ42-expressing flies ( p

    Article Snippet: Plates were fixed with 4% PFA (v/v) for 15 minutes, washed three times with PBS, permeabilized with 0.5% Triton-X before staining with primary antibodies against Keap1 (1:50; ab150654, Abcam) and Nrf2 (1:100; ab62352, Abcam) in blocking solution containing 10% goat serum and 3% BSA overnight at 4 C. After 3 washes in PBS secondary antibodies (α rabbit Alexa 594, ab150080, α mouse FITC, ab6785, Abcam) were applied.

    Techniques: Inhibition, Expressing

    Keap1 and GSK-3 in the regulation of Nrf2 in Alzheimer’s disease. (A) Aβ42 peptide inhibits activity of Nrf2, and this may explain the increased presence of xenobiotic and oxidative stress markers observed in Alzheimer’s disease. (B) Although lithium can activate Nrf2 at high concentrations, its protective effect against Aβ42 toxicity appears to be mainly Nrf2-independent, reducing Aβ42 levels by inhibiting translation[ 47 ] and preventing oxidative damage. More specific GSK-3 inhibitors are required to confirm the precise role of GSK-3 in rescuing Nrf2 deficits in neurodegenerative disease. (C) Genetic and pharmacological inhibition of Keap1 can rescue Aβ42-induced Nrf2 inhibition and neuronal toxicity by preventing xenobiotic damage and activating degradation of Aβ42 peptide. (D) Keap1 inhibitors may serve as effective therapies for AD and, in combination with GSK-3 inhibitors, may provide added benefits in preventing neurodegeneration through non-overlapping mechanisms.

    Journal: PLoS Genetics

    Article Title: Direct Keap1-Nrf2 disruption as a potential therapeutic target for Alzheimer’s disease

    doi: 10.1371/journal.pgen.1006593

    Figure Lengend Snippet: Keap1 and GSK-3 in the regulation of Nrf2 in Alzheimer’s disease. (A) Aβ42 peptide inhibits activity of Nrf2, and this may explain the increased presence of xenobiotic and oxidative stress markers observed in Alzheimer’s disease. (B) Although lithium can activate Nrf2 at high concentrations, its protective effect against Aβ42 toxicity appears to be mainly Nrf2-independent, reducing Aβ42 levels by inhibiting translation[ 47 ] and preventing oxidative damage. More specific GSK-3 inhibitors are required to confirm the precise role of GSK-3 in rescuing Nrf2 deficits in neurodegenerative disease. (C) Genetic and pharmacological inhibition of Keap1 can rescue Aβ42-induced Nrf2 inhibition and neuronal toxicity by preventing xenobiotic damage and activating degradation of Aβ42 peptide. (D) Keap1 inhibitors may serve as effective therapies for AD and, in combination with GSK-3 inhibitors, may provide added benefits in preventing neurodegeneration through non-overlapping mechanisms.

    Article Snippet: Plates were fixed with 4% PFA (v/v) for 15 minutes, washed three times with PBS, permeabilized with 0.5% Triton-X before staining with primary antibodies against Keap1 (1:50; ab150654, Abcam) and Nrf2 (1:100; ab62352, Abcam) in blocking solution containing 10% goat serum and 3% BSA overnight at 4 C. After 3 washes in PBS secondary antibodies (α rabbit Alexa 594, ab150080, α mouse FITC, ab6785, Abcam) were applied.

    Techniques: Activity Assay, Inhibition

    Direct Keap1-Nrf2 inhibitors protect mouse neurons from Aβ toxicity. (A) Molecular structure of compound 22h (1-(3-methylphenyl)-4-(3-nitrophenyl)-1,2,3-triazole[ 22 ]. (B) Aβ oligomer toxicity, as measured by sensitivity to resazurin (see methods ), in SH-SY5Y cells treated with a 50% dilution of either WT or 7PA2 CHO cell conditioned medium (** p

    Journal: PLoS Genetics

    Article Title: Direct Keap1-Nrf2 disruption as a potential therapeutic target for Alzheimer’s disease

    doi: 10.1371/journal.pgen.1006593

    Figure Lengend Snippet: Direct Keap1-Nrf2 inhibitors protect mouse neurons from Aβ toxicity. (A) Molecular structure of compound 22h (1-(3-methylphenyl)-4-(3-nitrophenyl)-1,2,3-triazole[ 22 ]. (B) Aβ oligomer toxicity, as measured by sensitivity to resazurin (see methods ), in SH-SY5Y cells treated with a 50% dilution of either WT or 7PA2 CHO cell conditioned medium (** p

    Article Snippet: Plates were fixed with 4% PFA (v/v) for 15 minutes, washed three times with PBS, permeabilized with 0.5% Triton-X before staining with primary antibodies against Keap1 (1:50; ab150654, Abcam) and Nrf2 (1:100; ab62352, Abcam) in blocking solution containing 10% goat serum and 3% BSA overnight at 4 C. After 3 washes in PBS secondary antibodies (α rabbit Alexa 594, ab150080, α mouse FITC, ab6785, Abcam) were applied.

    Techniques:

    Keap1 reduction, but not lithium treatment, protects against Aβ42 toxicity in correlation with increased cncC activity. (A) Heterozygous loss of Keap1 extended lifespan of ArcAβ42-expressing flies. P = 0.001 comparing +RU, Keap1 del or +RU, Keap1 EY5 flies to +RU alone (log-rank test). N = 150 flies per condition. (B) Heterozygous loss of Keap1 ameliorated climbing deficiency in ArcAβ42 flies. P

    Journal: PLoS Genetics

    Article Title: Direct Keap1-Nrf2 disruption as a potential therapeutic target for Alzheimer’s disease

    doi: 10.1371/journal.pgen.1006593

    Figure Lengend Snippet: Keap1 reduction, but not lithium treatment, protects against Aβ42 toxicity in correlation with increased cncC activity. (A) Heterozygous loss of Keap1 extended lifespan of ArcAβ42-expressing flies. P = 0.001 comparing +RU, Keap1 del or +RU, Keap1 EY5 flies to +RU alone (log-rank test). N = 150 flies per condition. (B) Heterozygous loss of Keap1 ameliorated climbing deficiency in ArcAβ42 flies. P

    Article Snippet: Plates were fixed with 4% PFA (v/v) for 15 minutes, washed three times with PBS, permeabilized with 0.5% Triton-X before staining with primary antibodies against Keap1 (1:50; ab150654, Abcam) and Nrf2 (1:100; ab62352, Abcam) in blocking solution containing 10% goat serum and 3% BSA overnight at 4 C. After 3 washes in PBS secondary antibodies (α rabbit Alexa 594, ab150080, α mouse FITC, ab6785, Abcam) were applied.

    Techniques: Activity Assay, Expressing

    Keap1 inhibition enhances degradation of the Aβ42 peptide. (A) Lowering Keap1 reduced total Aβ42 peptide levels in 14 day-old flies. Data are presented as means ± SEM. P

    Journal: PLoS Genetics

    Article Title: Direct Keap1-Nrf2 disruption as a potential therapeutic target for Alzheimer’s disease

    doi: 10.1371/journal.pgen.1006593

    Figure Lengend Snippet: Keap1 inhibition enhances degradation of the Aβ42 peptide. (A) Lowering Keap1 reduced total Aβ42 peptide levels in 14 day-old flies. Data are presented as means ± SEM. P

    Article Snippet: Plates were fixed with 4% PFA (v/v) for 15 minutes, washed three times with PBS, permeabilized with 0.5% Triton-X before staining with primary antibodies against Keap1 (1:50; ab150654, Abcam) and Nrf2 (1:100; ab62352, Abcam) in blocking solution containing 10% goat serum and 3% BSA overnight at 4 C. After 3 washes in PBS secondary antibodies (α rabbit Alexa 594, ab150080, α mouse FITC, ab6785, Abcam) were applied.

    Techniques: Inhibition

    Nrf2 and Keap1 immunohistochemical stains in normal esophageal mucosae and esophageal squamous cell carcinoma (ESCC). ( a ) Representative cases of Nrf2 staining. Neither cytoplasmic nor nuclear expression of Nrf2 was common in normal esophageal mucosa (intensity = 0). ESCC samples displayed increased Nrf2 staining in both the cytoplasm and nucleus. ( b ) Representative cases of Keap1 staining. Keap1 expression was high in the normal esophageal mucosa (intensity = 3). ESCC showed various staining patterns of Keap1 and limited expression was common. (Original magnification = 400,Scale bar 50 μm). Black arrows indicate positive nuclear staining and white arrows indicate positive cytoplasmic staining. ( c ) Comparison of immunohistochemical Q scores of Nrf2 and Keap1 between ESCC and normal esophageal mucosae. The medium lines of boxes show the median value, the top and bottom lines of boxes represent the 75th and 25th percentiles, respectively; and the ends of whiskers represent the 10th and 90th percentiles.

    Journal: Thoracic Cancer

    Article Title: Nrf2 and Keap1 abnormalities in esophageal squamous cell carcinoma and association with the effect of chemoradiotherapy

    doi: 10.1111/1759-7714.12640

    Figure Lengend Snippet: Nrf2 and Keap1 immunohistochemical stains in normal esophageal mucosae and esophageal squamous cell carcinoma (ESCC). ( a ) Representative cases of Nrf2 staining. Neither cytoplasmic nor nuclear expression of Nrf2 was common in normal esophageal mucosa (intensity = 0). ESCC samples displayed increased Nrf2 staining in both the cytoplasm and nucleus. ( b ) Representative cases of Keap1 staining. Keap1 expression was high in the normal esophageal mucosa (intensity = 3). ESCC showed various staining patterns of Keap1 and limited expression was common. (Original magnification = 400,Scale bar 50 μm). Black arrows indicate positive nuclear staining and white arrows indicate positive cytoplasmic staining. ( c ) Comparison of immunohistochemical Q scores of Nrf2 and Keap1 between ESCC and normal esophageal mucosae. The medium lines of boxes show the median value, the top and bottom lines of boxes represent the 75th and 25th percentiles, respectively; and the ends of whiskers represent the 10th and 90th percentiles.

    Article Snippet: The sections were soaked in 3% hydrogen peroxide solution for 10 minutes to reduce endogenous peroxidase activity and were incubated afterward with primary antibodies against Nrf2 (ab31163, Abcam, Cambridge, UK) and Keap1 (10503‐2‐AP, Proteintech, Chicago, IL, USA) for two hours at room temperature.

    Techniques: Immunohistochemistry, Staining, Expressing

    Receiver operating characteristic curve for prediction of esophageal squamous cell carcinoma using immunohistochemical Q scores of Nrf2 and Keap1. The area under the curves (AUC) of nuclear Nrf2, cytoplasmic Nrf2, and Keap1 Q scores were 0.829, 0.682, and 0.619, respectively. CI, confidence interval.

    Journal: Thoracic Cancer

    Article Title: Nrf2 and Keap1 abnormalities in esophageal squamous cell carcinoma and association with the effect of chemoradiotherapy

    doi: 10.1111/1759-7714.12640

    Figure Lengend Snippet: Receiver operating characteristic curve for prediction of esophageal squamous cell carcinoma using immunohistochemical Q scores of Nrf2 and Keap1. The area under the curves (AUC) of nuclear Nrf2, cytoplasmic Nrf2, and Keap1 Q scores were 0.829, 0.682, and 0.619, respectively. CI, confidence interval.

    Article Snippet: The sections were soaked in 3% hydrogen peroxide solution for 10 minutes to reduce endogenous peroxidase activity and were incubated afterward with primary antibodies against Nrf2 (ab31163, Abcam, Cambridge, UK) and Keap1 (10503‐2‐AP, Proteintech, Chicago, IL, USA) for two hours at room temperature.

    Techniques: Immunohistochemistry

    Manipulation of SQSTM1 levels affects the basal protein level of Keap1 in a panel of human cell lines. A , HeLa, HEK293T, HepG2, Huh-7 and CFPAC-1 cells were transfected with a scrambled control siRNA (si-CON) or SQSTM1-targeting siRNA (si-SQSTM1 h3) for 48 h. Keap1 and SQSTM1 levels were analyzed by Western blotting. Immunoreactive band volumes were quantified by densitometry and expressed relative to β-actin to enable comparison of Keap1 protein levels in cells transfected with si-CON, which were arbitrarily set at 1.00, and those transfected with si-SQSTM1 h3. Relative Keap1 levels are presented beneath the Keap1 blot. B , HeLa, HEK293T, and HepG2 cells were mock transfected or transfected with a SQSTM1-FLAG/His construct for 48 h. Keap1 and SQSTM1 levels were analyzed by Western blotting. Immunoreactive band volumes were quantified by densitometry and expressed relative to β-actin to enable comparison of Keap1 protein levels in mock transfected cells, which were arbitrarily set at 1.00, and those transfected with SQSTM1-FLAG/His. Relative Keap1 levels are presented beneath the Keap1 blot.

    Journal: The Journal of Biological Chemistry

    Article Title: Physical and Functional Interaction of Sequestosome 1 with Keap1 Regulates the Keap1-Nrf2 Cell Defense Pathway *

    doi: 10.1074/jbc.M109.096545

    Figure Lengend Snippet: Manipulation of SQSTM1 levels affects the basal protein level of Keap1 in a panel of human cell lines. A , HeLa, HEK293T, HepG2, Huh-7 and CFPAC-1 cells were transfected with a scrambled control siRNA (si-CON) or SQSTM1-targeting siRNA (si-SQSTM1 h3) for 48 h. Keap1 and SQSTM1 levels were analyzed by Western blotting. Immunoreactive band volumes were quantified by densitometry and expressed relative to β-actin to enable comparison of Keap1 protein levels in cells transfected with si-CON, which were arbitrarily set at 1.00, and those transfected with si-SQSTM1 h3. Relative Keap1 levels are presented beneath the Keap1 blot. B , HeLa, HEK293T, and HepG2 cells were mock transfected or transfected with a SQSTM1-FLAG/His construct for 48 h. Keap1 and SQSTM1 levels were analyzed by Western blotting. Immunoreactive band volumes were quantified by densitometry and expressed relative to β-actin to enable comparison of Keap1 protein levels in mock transfected cells, which were arbitrarily set at 1.00, and those transfected with SQSTM1-FLAG/His. Relative Keap1 levels are presented beneath the Keap1 blot.

    Article Snippet: SQSTM1 Influences the Basal Integrity of the Keap1-Nrf2 Pathway by Regulating the Degradation of Keap1 To investigate the biological significance of the interaction between Keap1 and SQSTM1, we examined the effect on Keap1 of depleting endogenous SQSTM1, using siRNA, or ectopically expressing SQSTM1-FLAG/His in a panel of human cell lines originating from discrete organs.

    Techniques: Transfection, Western Blot, Construct

    SQSTM1 associates with Keap1 in mammalian cells. A , overview of proteomic approach used to identify SQSTM1 as a binding partner of Keap1. HEK293T cells were transfected with Keap1-V5/His, which was purified, by anti-V5 pulldown, from cell lysates, reduced, alkylated, and digested overnight with trypsin. The resulting tryptic peptides were analyzed by LC-ESI-MS/MS. Proteins present in the immunopurified fraction were identified via reference to the expected masses of their tryptic peptides. The MS/MS spectrum depicts the SQSTM1 peptide 268 LTPVSPESSSTEEK 281 , [M+2H] 2+ = 745.9 atomic mass units. y- and b-ions are labeled where present. Immonium ions are labeled with the one-letter code for their corresponding amino acid. DTT , dithiothreitol; NEM , N -ethylmaleimide. B , Keap1-V5/His or SQSTM1-FLAG/His were ectopically expressed in HEK293T or HeLa cells. Lysates were prepared and incubated with anti-V5 or anti-FLAG antibody-conjugated agarose beads, and immunopurified Keap1 and SQSTM1 were analyzed by Western blotting. Input represents 5% of the total cell material preimmunopurification. β-Actin was probed as a loading control. C , HEK293T cells were lysed and subjected to immunoprecipitation ( IP ) with an anti-SQSTM1 antibody and protein A-conjugated beads. A similar procedure was performed without the anti-SQSTM1 antibody as a control. Lysates and immunoprecipitated proteins were analyzed by Western blotting. Lysates before and after immunoprecipitation represent 5% of the total cell material. Lysates from HEK293T cells in which SQSTM1 and Keap1 had been ectopically expressed were loaded as positive controls. The inherent immunoreactivity of protein A-Sepharose was also determined as a control.

    Journal: The Journal of Biological Chemistry

    Article Title: Physical and Functional Interaction of Sequestosome 1 with Keap1 Regulates the Keap1-Nrf2 Cell Defense Pathway *

    doi: 10.1074/jbc.M109.096545

    Figure Lengend Snippet: SQSTM1 associates with Keap1 in mammalian cells. A , overview of proteomic approach used to identify SQSTM1 as a binding partner of Keap1. HEK293T cells were transfected with Keap1-V5/His, which was purified, by anti-V5 pulldown, from cell lysates, reduced, alkylated, and digested overnight with trypsin. The resulting tryptic peptides were analyzed by LC-ESI-MS/MS. Proteins present in the immunopurified fraction were identified via reference to the expected masses of their tryptic peptides. The MS/MS spectrum depicts the SQSTM1 peptide 268 LTPVSPESSSTEEK 281 , [M+2H] 2+ = 745.9 atomic mass units. y- and b-ions are labeled where present. Immonium ions are labeled with the one-letter code for their corresponding amino acid. DTT , dithiothreitol; NEM , N -ethylmaleimide. B , Keap1-V5/His or SQSTM1-FLAG/His were ectopically expressed in HEK293T or HeLa cells. Lysates were prepared and incubated with anti-V5 or anti-FLAG antibody-conjugated agarose beads, and immunopurified Keap1 and SQSTM1 were analyzed by Western blotting. Input represents 5% of the total cell material preimmunopurification. β-Actin was probed as a loading control. C , HEK293T cells were lysed and subjected to immunoprecipitation ( IP ) with an anti-SQSTM1 antibody and protein A-conjugated beads. A similar procedure was performed without the anti-SQSTM1 antibody as a control. Lysates and immunoprecipitated proteins were analyzed by Western blotting. Lysates before and after immunoprecipitation represent 5% of the total cell material. Lysates from HEK293T cells in which SQSTM1 and Keap1 had been ectopically expressed were loaded as positive controls. The inherent immunoreactivity of protein A-Sepharose was also determined as a control.

    Article Snippet: SQSTM1 Influences the Basal Integrity of the Keap1-Nrf2 Pathway by Regulating the Degradation of Keap1 To investigate the biological significance of the interaction between Keap1 and SQSTM1, we examined the effect on Keap1 of depleting endogenous SQSTM1, using siRNA, or ectopically expressing SQSTM1-FLAG/His in a panel of human cell lines originating from discrete organs.

    Techniques: Binding Assay, Transfection, Purification, Mass Spectrometry, Labeling, Incubation, Western Blot, Immunoprecipitation

    Model for the role of SQSTM1 in regulating the integrity of the Keap1-Nrf2 cell defense pathway. This study has demonstrated that SQSTM1 associates with Keap1 in cells and contributes to the integrity of the Keap1-Nrf2 pathway by regulating the basal levels of Keap1 and Nrf2. Recent work has demonstrated increased levels of Keap1 in autophagy-deficient cells ( 23 ). SQSTM1 expression is itself up-regulated in an Nrf2-dependent manner under conditions of chemical/oxidative stress ( 27 , 28 ), suggesting that SQSTM1 forms part of a regulatory feedback loop in the Keap1-Nrf2 pathway. Under conditions of Nrf2 activation, the elevated expression of SQSTM1 may enable the targeting of Keap1 for degradation via autophagy, which in turn may (i) further contribute to the activation of Nrf2 and/or (ii) represent a means by which modified/inactivated Keap1 is removed/recycled to restore homeostasis in the pathway. ARE/EpRE , antioxidant response element/electrophile response element.

    Journal: The Journal of Biological Chemistry

    Article Title: Physical and Functional Interaction of Sequestosome 1 with Keap1 Regulates the Keap1-Nrf2 Cell Defense Pathway *

    doi: 10.1074/jbc.M109.096545

    Figure Lengend Snippet: Model for the role of SQSTM1 in regulating the integrity of the Keap1-Nrf2 cell defense pathway. This study has demonstrated that SQSTM1 associates with Keap1 in cells and contributes to the integrity of the Keap1-Nrf2 pathway by regulating the basal levels of Keap1 and Nrf2. Recent work has demonstrated increased levels of Keap1 in autophagy-deficient cells ( 23 ). SQSTM1 expression is itself up-regulated in an Nrf2-dependent manner under conditions of chemical/oxidative stress ( 27 , 28 ), suggesting that SQSTM1 forms part of a regulatory feedback loop in the Keap1-Nrf2 pathway. Under conditions of Nrf2 activation, the elevated expression of SQSTM1 may enable the targeting of Keap1 for degradation via autophagy, which in turn may (i) further contribute to the activation of Nrf2 and/or (ii) represent a means by which modified/inactivated Keap1 is removed/recycled to restore homeostasis in the pathway. ARE/EpRE , antioxidant response element/electrophile response element.

    Article Snippet: SQSTM1 Influences the Basal Integrity of the Keap1-Nrf2 Pathway by Regulating the Degradation of Keap1 To investigate the biological significance of the interaction between Keap1 and SQSTM1, we examined the effect on Keap1 of depleting endogenous SQSTM1, using siRNA, or ectopically expressing SQSTM1-FLAG/His in a panel of human cell lines originating from discrete organs.

    Techniques: Expressing, Activation Assay, Modification

    Depletion of SQSTM1 affects the basal protein levels of Keap1 and Nrf2, through a decrease in the rate of degradation of Keap1, but does not compromise the inducibility of Nrf2. Hepa-1c1c7 cells were transfected with a scrambled control siRNA (si-CON) or SQSTM1-targetting siRNA (si-SQSTM1 m3 or m4) for 48 h. A , immunocytochemical analysis of total cellular SQSTM1 ( green ) levels following transfection with si-CON or si-SQSTM1 m3, as visualized by confocal microscopy. Nuclei were counterstained with Hoechst 33258 ( blue ). Each image represents an overlay of SQSTM1 and Hoechst signals. Scale bar , 50 μm. B , Western blot analysis of Keap1, Nrf2, and SQSTM1 in siRNA-transfected cells. Immunoreactive band volumes were quantified by densitometry and expressed relative to β-actin to enable comparison of Keap1 and Nrf2 protein levels in cells transfected with si-CON, which were arbitrarily set at 1.00, and those transfected with si-SQSTM1 m3 or m4. Relative Keap1 and Nrf2 levels are presented beneath the respective blots. C , relative levels of Keap1 and Nrf2 mRNA in siRNA-transfected cells, as determined by real-time RT-PCR using SYBR Green and gene-specific primers. Keap1 and Nrf2 mRNA levels were normalized to GAPDH mRNA levels in the same samples. The mRNA levels in si-SQSTM1-transfected cells are expressed relative to those detected in si-CON-transfected cells. D , siRNA-transfected cells exposed to cycloheximide ( CHX ; 35 μ m ) for the indicated times. Keap1 and SQSTM1 levels were analyzed by Western blotting. Immunoreactive band volumes were quantified by densitometry and expressed relative to β-actin to enable comparison of the Keap1 protein level at 0 h, which was arbitrarily set at 1.00, with that at subsequent time points. Relative levels of Keap1 are presented in the scatterplot. E , Western blot analysis of Nrf2 and SQSTM1 following exposure of siRNA-transfected cells to the Nrf2-activators tert -butylhydroquinone ( tBHQ ) or iodoacetamide (both 50 μ m ) for 1 h. Immunoreactive band volumes were quantified by densitometry and expressed relative to β-actin to enable comparison of Nrf2 protein levels in vehicle-exposed cells transfected with si-CON a or si-SQSTM1 m3 b , which were arbitrarily set at 1.00, and those exposed to tert -butylhydroquinone or iodoacetamide. Relative Nrf2 levels are presented beneath the Nrf2 blot.

    Journal: The Journal of Biological Chemistry

    Article Title: Physical and Functional Interaction of Sequestosome 1 with Keap1 Regulates the Keap1-Nrf2 Cell Defense Pathway *

    doi: 10.1074/jbc.M109.096545

    Figure Lengend Snippet: Depletion of SQSTM1 affects the basal protein levels of Keap1 and Nrf2, through a decrease in the rate of degradation of Keap1, but does not compromise the inducibility of Nrf2. Hepa-1c1c7 cells were transfected with a scrambled control siRNA (si-CON) or SQSTM1-targetting siRNA (si-SQSTM1 m3 or m4) for 48 h. A , immunocytochemical analysis of total cellular SQSTM1 ( green ) levels following transfection with si-CON or si-SQSTM1 m3, as visualized by confocal microscopy. Nuclei were counterstained with Hoechst 33258 ( blue ). Each image represents an overlay of SQSTM1 and Hoechst signals. Scale bar , 50 μm. B , Western blot analysis of Keap1, Nrf2, and SQSTM1 in siRNA-transfected cells. Immunoreactive band volumes were quantified by densitometry and expressed relative to β-actin to enable comparison of Keap1 and Nrf2 protein levels in cells transfected with si-CON, which were arbitrarily set at 1.00, and those transfected with si-SQSTM1 m3 or m4. Relative Keap1 and Nrf2 levels are presented beneath the respective blots. C , relative levels of Keap1 and Nrf2 mRNA in siRNA-transfected cells, as determined by real-time RT-PCR using SYBR Green and gene-specific primers. Keap1 and Nrf2 mRNA levels were normalized to GAPDH mRNA levels in the same samples. The mRNA levels in si-SQSTM1-transfected cells are expressed relative to those detected in si-CON-transfected cells. D , siRNA-transfected cells exposed to cycloheximide ( CHX ; 35 μ m ) for the indicated times. Keap1 and SQSTM1 levels were analyzed by Western blotting. Immunoreactive band volumes were quantified by densitometry and expressed relative to β-actin to enable comparison of the Keap1 protein level at 0 h, which was arbitrarily set at 1.00, with that at subsequent time points. Relative levels of Keap1 are presented in the scatterplot. E , Western blot analysis of Nrf2 and SQSTM1 following exposure of siRNA-transfected cells to the Nrf2-activators tert -butylhydroquinone ( tBHQ ) or iodoacetamide (both 50 μ m ) for 1 h. Immunoreactive band volumes were quantified by densitometry and expressed relative to β-actin to enable comparison of Nrf2 protein levels in vehicle-exposed cells transfected with si-CON a or si-SQSTM1 m3 b , which were arbitrarily set at 1.00, and those exposed to tert -butylhydroquinone or iodoacetamide. Relative Nrf2 levels are presented beneath the Nrf2 blot.

    Article Snippet: SQSTM1 Influences the Basal Integrity of the Keap1-Nrf2 Pathway by Regulating the Degradation of Keap1 To investigate the biological significance of the interaction between Keap1 and SQSTM1, we examined the effect on Keap1 of depleting endogenous SQSTM1, using siRNA, or ectopically expressing SQSTM1-FLAG/His in a panel of human cell lines originating from discrete organs.

    Techniques: Transfection, Confocal Microscopy, Western Blot, Quantitative RT-PCR, SYBR Green Assay

    Silencing of Keap1 in the liver by siRNA administration in vivo reduces ConA-mediated apoptosis. Analysis of cell-death markers in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A) TNFα mRNA levels determined by real-time PCR. Data are presented as mean±s.e.m. relative to siLuc mice. (B) Representative blots with the indicated antibodies. (C) Graphics of caspase 3 and 8 enzymatic activities. Data are presented as mean±s.e.m. relative to siLuc mice. (D) Representative blots with the indicated antibodies. * P

    Journal: Disease Models & Mechanisms

    Article Title: In vivo siRNA delivery of Keap1 modulates death and survival signaling pathways and attenuates concanavalin-A-induced acute liver injury in mice

    doi: 10.1242/dmm.015537

    Figure Lengend Snippet: Silencing of Keap1 in the liver by siRNA administration in vivo reduces ConA-mediated apoptosis. Analysis of cell-death markers in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A) TNFα mRNA levels determined by real-time PCR. Data are presented as mean±s.e.m. relative to siLuc mice. (B) Representative blots with the indicated antibodies. (C) Graphics of caspase 3 and 8 enzymatic activities. Data are presented as mean±s.e.m. relative to siLuc mice. (D) Representative blots with the indicated antibodies. * P

    Article Snippet: Antibodies The antibodies used were: anti-phospho JNK (Thr183/Tyr185) (#4668), anti-phospho STAT3 (Tyr705) (#9131), anti-STAT3 (#8719), anti-phospho p38 MAPK (Thr180/Tyr182) (#9211), anti-p38 MAPK (#9212), anti-phospho Foxo1 (#9461) and anti-Akt (#9272) from Cell Signaling Technology (MA, USA); anti-phospho IGFIR (Tyr1165/1166) (sc-101704), anti-JNK (sc-571), anti-phospho-Akt1/2/3 (Ser473) (sc-7985-R), anti-caspase 1 (sc-514), anti-Nrf2 (sc-722) and anti-Keap1 (sc-33569) from Santa Cruz (Palo Alto, CA); anti-phospho IRS1 (Tyr1179) (07-844), anti-phospho IRS1 (Ser 307) (07-247), anti-IRS1 (06-248), anti-p85α (06-195) and anti-HO1 (AB1284) antibodies from Merck Millipore (Merck KGaA, Darmstadt, Germany); anti-β-actin (A-5441) antibody from Sigma Chemical Co. (St Louis, MO); anti-Lamin B (aB16048) and FasL (aB68338) from Abcam (Abcam, Cambridge, UK).

    Techniques: In Vivo, Luciferase, Mouse Assay, Real-time Polymerase Chain Reaction

    Reduction of Keap1 in the liver by siRNA administration in vivo attenuates ConA-induced liver damage. Analysis of Keap1 expression and histopathological features in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A) (left panel) Keap1 mRNA levels determined by real-time PCR. (Right panel) Representative blots with the indicated antibodies and quantification of the densitometric analysis from all blots. Data are presented as mean±s.e.m. relative to siLuc mice. (B) Cbr3 and Nqo1 mRNA levels determined by real-time PCR. Data are presented as mean±s.e.m. relative to siLuc mice. (C) Representative images from hematoxylin and eosin staining. *** P

    Journal: Disease Models & Mechanisms

    Article Title: In vivo siRNA delivery of Keap1 modulates death and survival signaling pathways and attenuates concanavalin-A-induced acute liver injury in mice

    doi: 10.1242/dmm.015537

    Figure Lengend Snippet: Reduction of Keap1 in the liver by siRNA administration in vivo attenuates ConA-induced liver damage. Analysis of Keap1 expression and histopathological features in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A) (left panel) Keap1 mRNA levels determined by real-time PCR. (Right panel) Representative blots with the indicated antibodies and quantification of the densitometric analysis from all blots. Data are presented as mean±s.e.m. relative to siLuc mice. (B) Cbr3 and Nqo1 mRNA levels determined by real-time PCR. Data are presented as mean±s.e.m. relative to siLuc mice. (C) Representative images from hematoxylin and eosin staining. *** P

    Article Snippet: Antibodies The antibodies used were: anti-phospho JNK (Thr183/Tyr185) (#4668), anti-phospho STAT3 (Tyr705) (#9131), anti-STAT3 (#8719), anti-phospho p38 MAPK (Thr180/Tyr182) (#9211), anti-p38 MAPK (#9212), anti-phospho Foxo1 (#9461) and anti-Akt (#9272) from Cell Signaling Technology (MA, USA); anti-phospho IGFIR (Tyr1165/1166) (sc-101704), anti-JNK (sc-571), anti-phospho-Akt1/2/3 (Ser473) (sc-7985-R), anti-caspase 1 (sc-514), anti-Nrf2 (sc-722) and anti-Keap1 (sc-33569) from Santa Cruz (Palo Alto, CA); anti-phospho IRS1 (Tyr1179) (07-844), anti-phospho IRS1 (Ser 307) (07-247), anti-IRS1 (06-248), anti-p85α (06-195) and anti-HO1 (AB1284) antibodies from Merck Millipore (Merck KGaA, Darmstadt, Germany); anti-β-actin (A-5441) antibody from Sigma Chemical Co. (St Louis, MO); anti-Lamin B (aB16048) and FasL (aB68338) from Abcam (Abcam, Cambridge, UK).

    Techniques: In Vivo, Expressing, Luciferase, Mouse Assay, Real-time Polymerase Chain Reaction, Staining

    Silencing of Keap1 reduced the expression of inflammatory markers in ConA-injected livers. Analysis of inflammatory markers in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A,B) mRNA levels of the indicated cytokines and pro-inflammatory markers determined by real-time PCR. Data are presented as mean±s.e.m. relative to siLuc mice. * P

    Journal: Disease Models & Mechanisms

    Article Title: In vivo siRNA delivery of Keap1 modulates death and survival signaling pathways and attenuates concanavalin-A-induced acute liver injury in mice

    doi: 10.1242/dmm.015537

    Figure Lengend Snippet: Silencing of Keap1 reduced the expression of inflammatory markers in ConA-injected livers. Analysis of inflammatory markers in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A,B) mRNA levels of the indicated cytokines and pro-inflammatory markers determined by real-time PCR. Data are presented as mean±s.e.m. relative to siLuc mice. * P

    Article Snippet: Antibodies The antibodies used were: anti-phospho JNK (Thr183/Tyr185) (#4668), anti-phospho STAT3 (Tyr705) (#9131), anti-STAT3 (#8719), anti-phospho p38 MAPK (Thr180/Tyr182) (#9211), anti-p38 MAPK (#9212), anti-phospho Foxo1 (#9461) and anti-Akt (#9272) from Cell Signaling Technology (MA, USA); anti-phospho IGFIR (Tyr1165/1166) (sc-101704), anti-JNK (sc-571), anti-phospho-Akt1/2/3 (Ser473) (sc-7985-R), anti-caspase 1 (sc-514), anti-Nrf2 (sc-722) and anti-Keap1 (sc-33569) from Santa Cruz (Palo Alto, CA); anti-phospho IRS1 (Tyr1179) (07-844), anti-phospho IRS1 (Ser 307) (07-247), anti-IRS1 (06-248), anti-p85α (06-195) and anti-HO1 (AB1284) antibodies from Merck Millipore (Merck KGaA, Darmstadt, Germany); anti-β-actin (A-5441) antibody from Sigma Chemical Co. (St Louis, MO); anti-Lamin B (aB16048) and FasL (aB68338) from Abcam (Abcam, Cambridge, UK).

    Techniques: Expressing, Injection, Luciferase, Mouse Assay, Real-time Polymerase Chain Reaction

    ConA-induced oxidative stress is reduced in livers from mice injected with Keap1 siRNA. Analysis of Nrf2 signaling and oxidative stress markers in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A,B) Representative blots with the indicated antibodies. (C) Keap1 mRNA levels determined by real-time PCR. Data are presented as mean±s.e.m. relative to siLuc mice. (D) Graphic of carbonylated protein levels. Data are presented as mean±s.e.m. relative to siLuc mice. ** P

    Journal: Disease Models & Mechanisms

    Article Title: In vivo siRNA delivery of Keap1 modulates death and survival signaling pathways and attenuates concanavalin-A-induced acute liver injury in mice

    doi: 10.1242/dmm.015537

    Figure Lengend Snippet: ConA-induced oxidative stress is reduced in livers from mice injected with Keap1 siRNA. Analysis of Nrf2 signaling and oxidative stress markers in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A,B) Representative blots with the indicated antibodies. (C) Keap1 mRNA levels determined by real-time PCR. Data are presented as mean±s.e.m. relative to siLuc mice. (D) Graphic of carbonylated protein levels. Data are presented as mean±s.e.m. relative to siLuc mice. ** P

    Article Snippet: Antibodies The antibodies used were: anti-phospho JNK (Thr183/Tyr185) (#4668), anti-phospho STAT3 (Tyr705) (#9131), anti-STAT3 (#8719), anti-phospho p38 MAPK (Thr180/Tyr182) (#9211), anti-p38 MAPK (#9212), anti-phospho Foxo1 (#9461) and anti-Akt (#9272) from Cell Signaling Technology (MA, USA); anti-phospho IGFIR (Tyr1165/1166) (sc-101704), anti-JNK (sc-571), anti-phospho-Akt1/2/3 (Ser473) (sc-7985-R), anti-caspase 1 (sc-514), anti-Nrf2 (sc-722) and anti-Keap1 (sc-33569) from Santa Cruz (Palo Alto, CA); anti-phospho IRS1 (Tyr1179) (07-844), anti-phospho IRS1 (Ser 307) (07-247), anti-IRS1 (06-248), anti-p85α (06-195) and anti-HO1 (AB1284) antibodies from Merck Millipore (Merck KGaA, Darmstadt, Germany); anti-β-actin (A-5441) antibody from Sigma Chemical Co. (St Louis, MO); anti-Lamin B (aB16048) and FasL (aB68338) from Abcam (Abcam, Cambridge, UK).

    Techniques: Mouse Assay, Injection, Luciferase, Real-time Polymerase Chain Reaction

    Reduction of Keap1 preserved the IGFIR survival signaling pathway in ConA-injected livers. Analysis of IGFIR survival signaling in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A,B) Representative blots with the indicated antibodies and quantification of the densitometric analysis from all blots. Data are presented as mean±s.e.m. relative to siLuc mice. (C) PTP1B mRNA levels determined by real-time PCR. Data are presented as mean±s.e.m. relative to siLuc mice. * P

    Journal: Disease Models & Mechanisms

    Article Title: In vivo siRNA delivery of Keap1 modulates death and survival signaling pathways and attenuates concanavalin-A-induced acute liver injury in mice

    doi: 10.1242/dmm.015537

    Figure Lengend Snippet: Reduction of Keap1 preserved the IGFIR survival signaling pathway in ConA-injected livers. Analysis of IGFIR survival signaling in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A,B) Representative blots with the indicated antibodies and quantification of the densitometric analysis from all blots. Data are presented as mean±s.e.m. relative to siLuc mice. (C) PTP1B mRNA levels determined by real-time PCR. Data are presented as mean±s.e.m. relative to siLuc mice. * P

    Article Snippet: Antibodies The antibodies used were: anti-phospho JNK (Thr183/Tyr185) (#4668), anti-phospho STAT3 (Tyr705) (#9131), anti-STAT3 (#8719), anti-phospho p38 MAPK (Thr180/Tyr182) (#9211), anti-p38 MAPK (#9212), anti-phospho Foxo1 (#9461) and anti-Akt (#9272) from Cell Signaling Technology (MA, USA); anti-phospho IGFIR (Tyr1165/1166) (sc-101704), anti-JNK (sc-571), anti-phospho-Akt1/2/3 (Ser473) (sc-7985-R), anti-caspase 1 (sc-514), anti-Nrf2 (sc-722) and anti-Keap1 (sc-33569) from Santa Cruz (Palo Alto, CA); anti-phospho IRS1 (Tyr1179) (07-844), anti-phospho IRS1 (Ser 307) (07-247), anti-IRS1 (06-248), anti-p85α (06-195) and anti-HO1 (AB1284) antibodies from Merck Millipore (Merck KGaA, Darmstadt, Germany); anti-β-actin (A-5441) antibody from Sigma Chemical Co. (St Louis, MO); anti-Lamin B (aB16048) and FasL (aB68338) from Abcam (Abcam, Cambridge, UK).

    Techniques: Injection, Luciferase, Mouse Assay, Real-time Polymerase Chain Reaction

    Reduction of Keap1 decreased the activation of JNK in ConA-injected livers. Analysis of stress kinase activation in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A,B) Representative blots with the indicated antibodies.

    Journal: Disease Models & Mechanisms

    Article Title: In vivo siRNA delivery of Keap1 modulates death and survival signaling pathways and attenuates concanavalin-A-induced acute liver injury in mice

    doi: 10.1242/dmm.015537

    Figure Lengend Snippet: Reduction of Keap1 decreased the activation of JNK in ConA-injected livers. Analysis of stress kinase activation in liver samples from luciferase siRNA (siLuc) or Keap1 siRNA (siKeap1) mice after 4 and 8 hours of ConA treatment ( n =4–6 animals per condition). (A,B) Representative blots with the indicated antibodies.

    Article Snippet: Antibodies The antibodies used were: anti-phospho JNK (Thr183/Tyr185) (#4668), anti-phospho STAT3 (Tyr705) (#9131), anti-STAT3 (#8719), anti-phospho p38 MAPK (Thr180/Tyr182) (#9211), anti-p38 MAPK (#9212), anti-phospho Foxo1 (#9461) and anti-Akt (#9272) from Cell Signaling Technology (MA, USA); anti-phospho IGFIR (Tyr1165/1166) (sc-101704), anti-JNK (sc-571), anti-phospho-Akt1/2/3 (Ser473) (sc-7985-R), anti-caspase 1 (sc-514), anti-Nrf2 (sc-722) and anti-Keap1 (sc-33569) from Santa Cruz (Palo Alto, CA); anti-phospho IRS1 (Tyr1179) (07-844), anti-phospho IRS1 (Ser 307) (07-247), anti-IRS1 (06-248), anti-p85α (06-195) and anti-HO1 (AB1284) antibodies from Merck Millipore (Merck KGaA, Darmstadt, Germany); anti-β-actin (A-5441) antibody from Sigma Chemical Co. (St Louis, MO); anti-Lamin B (aB16048) and FasL (aB68338) from Abcam (Abcam, Cambridge, UK).

    Techniques: Activation Assay, Injection, Luciferase, Mouse Assay

    Effect of SKI-II on Keap1. A. Nuclear fractions from BEAS2B cells treated with SKI-II (1 µM) at increasing times (10–120 min) were analysed for Keap1 and Nrf2 expression and normalized using TBP (nuclear). Keap1 bands at 140 kDa and 69 kDa were analysed as fold change over non-treatment of the 69 kDa band. *** p

    Journal: PLoS ONE

    Article Title: Activation of Transcription Factor Nrf2 Signalling by the Sphingosine Kinase Inhibitor SKI-II Is Mediated by the Formation of Keap1 Dimers

    doi: 10.1371/journal.pone.0088168

    Figure Lengend Snippet: Effect of SKI-II on Keap1. A. Nuclear fractions from BEAS2B cells treated with SKI-II (1 µM) at increasing times (10–120 min) were analysed for Keap1 and Nrf2 expression and normalized using TBP (nuclear). Keap1 bands at 140 kDa and 69 kDa were analysed as fold change over non-treatment of the 69 kDa band. *** p

    Article Snippet: Antibodies against the following were used for immunoblotting: β-actin, p62, NQO1, ubiquitin (Sigma), Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA), Nrf2, GCLM, HO-1, TBP, β-actin, SK2 (Abcam, Cambridge, UK), SK1 (Abgent, San Diego, CA) and Keap1 (Origene, Rockville, MD).

    Techniques: Expressing

    Working model for the inhibitory of NOM on IL‐1β–induced inflammation and ECM degradation in mice chondrocytes by targeting the Keap1‐Nrf2 signalling. ECM, extracellular matrix; HO‐1, haem oxygenase‐1; IL, interleukin; Keap1, Kelch‐like ECH‐associated protein 1; NF‐κB, nuclear factor κB; NOM, nomilin; Nrf2, nuclear factor‐erythroid 2‐related factor‐2; OA, osteoarthritis

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Nomilin targets the Keap1‐Nrf2 signalling and ameliorates the development of osteoarthritis. Nomilin targets the Keap1‐Nrf2 signalling and ameliorates the development of osteoarthritis

    doi: 10.1111/jcmm.15484

    Figure Lengend Snippet: Working model for the inhibitory of NOM on IL‐1β–induced inflammation and ECM degradation in mice chondrocytes by targeting the Keap1‐Nrf2 signalling. ECM, extracellular matrix; HO‐1, haem oxygenase‐1; IL, interleukin; Keap1, Kelch‐like ECH‐associated protein 1; NF‐κB, nuclear factor κB; NOM, nomilin; Nrf2, nuclear factor‐erythroid 2‐related factor‐2; OA, osteoarthritis

    Article Snippet: The primary antibody against collagen Ⅱ, Lamin B1, iNOS, COX‐2 and GAPDH was acquired from Abcam, goat anti‐rabbit and antimouse IgG‐HRP were from Bioworld and antibodies against Keap1, Nrf2, HO‐1, COX‐2, IκBα and p65 were purchased from Cell Signaling Technology; Alexa Fluor® 488 labelled and Alexa Fluor®594 labelled Goat Anti‐Rabbit IgG (H + L) second antibody was purchased from Jackson ImmunoResearch.

    Techniques: Mouse Assay

    Effect of NOM on Keap1‐Nrf2 pathway. Chondrocytes were pre‐treated for 1 h with various concentrations of NOM (0, 5 and 10 μmol/L), followed by stimulation with or without IL‐1β (10 ng/mL) for 2 h. Nrf2, Keap1 and HO‐1 were determined by Western blot (A) and quantification analysis (B). Total proteins extracted were subjected to immunoprecipitation (IP) using specific antibodies against Keap1. Immunoprecipitates were then subjected to Western blot analyses using specific antibodies against Keap1 and Nrf2 (C). Quantitative analyses of immunoprecipitates relative to GAPDH by densitometry were conducted (D). After Nrf2 knock‐down, the protein expressions of Nrf2, HO‐1 and P65 in chondrocytes treated as above were visualized by Western blot (E) and quantified in (F). The production of MMP‐13 and collagen II was assessed by ELISA (G). The values are mean ± SD of five independent experiments. ** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Nomilin targets the Keap1‐Nrf2 signalling and ameliorates the development of osteoarthritis. Nomilin targets the Keap1‐Nrf2 signalling and ameliorates the development of osteoarthritis

    doi: 10.1111/jcmm.15484

    Figure Lengend Snippet: Effect of NOM on Keap1‐Nrf2 pathway. Chondrocytes were pre‐treated for 1 h with various concentrations of NOM (0, 5 and 10 μmol/L), followed by stimulation with or without IL‐1β (10 ng/mL) for 2 h. Nrf2, Keap1 and HO‐1 were determined by Western blot (A) and quantification analysis (B). Total proteins extracted were subjected to immunoprecipitation (IP) using specific antibodies against Keap1. Immunoprecipitates were then subjected to Western blot analyses using specific antibodies against Keap1 and Nrf2 (C). Quantitative analyses of immunoprecipitates relative to GAPDH by densitometry were conducted (D). After Nrf2 knock‐down, the protein expressions of Nrf2, HO‐1 and P65 in chondrocytes treated as above were visualized by Western blot (E) and quantified in (F). The production of MMP‐13 and collagen II was assessed by ELISA (G). The values are mean ± SD of five independent experiments. ** P

    Article Snippet: The primary antibody against collagen Ⅱ, Lamin B1, iNOS, COX‐2 and GAPDH was acquired from Abcam, goat anti‐rabbit and antimouse IgG‐HRP were from Bioworld and antibodies against Keap1, Nrf2, HO‐1, COX‐2, IκBα and p65 were purchased from Cell Signaling Technology; Alexa Fluor® 488 labelled and Alexa Fluor®594 labelled Goat Anti‐Rabbit IgG (H + L) second antibody was purchased from Jackson ImmunoResearch.

    Techniques: Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

    The messenger RNA (mRNA) levels of nuclear factor erythroid-2 like 2 (NRF2), heme-oxygenase1 (HMOX1), and kelch-like ECH-associated protein 1 (KEAP1) of diaphragm muscle. Data are expressed as mean ± standard deviation. A = p

    Journal: BioMed Research International

    Article Title: Exercise Prevents Diaphragm Wasting Induced by Cigarette Smoke through Modulation of Antioxidant Genes and Metalloproteinases

    doi: 10.1155/2018/5909053

    Figure Lengend Snippet: The messenger RNA (mRNA) levels of nuclear factor erythroid-2 like 2 (NRF2), heme-oxygenase1 (HMOX1), and kelch-like ECH-associated protein 1 (KEAP1) of diaphragm muscle. Data are expressed as mean ± standard deviation. A = p

    Article Snippet: Two microliters of each reverse transcription product were amplified in 20 L of 1X reaction buffer (TaqMan® Universal PCR Master Mix, Applied Biosystems) using appropriate TaqMan gene expression assays in a Step One Plus Thermocycler (Applied Biosystems) for the following genes: NRF2 (Mm00477784_m1); KEAP1 (Mm00497268_m1); and HMOX1 (Mm00516005_m1).

    Techniques: Standard Deviation

    Functional Consequences of E1-Keap1 Interaction ( a ) Subnetwork of interaction between HPV E1 and Keap1. Blue arrows denote the main contribution of differential mutation scores to KEAP1 through network propagation. MiST score of virus-human interaction in C33A cells is indicated (0.81). The mechanism of interaction is represented by edge shapes, with +Ub indicating ubiquitination. ( b , c , d , e ) Western Blot analysis of virus-host interaction by immunoprecipitation (IP) using streptavidin-coated beads to bind the Strep-tag ( b , d , e ) or an antibody against endogenous Keap1 ( c ). Proteins indicated on the right of each blot from IP and input samples were detected using the antibodies indicated. Bands were cropped from the same original membrane. Distinction between different strains of HPV is made using strain number preceding the respective viral bait (HPV-31 = 31, HPV-16 = 16; d ). ( f ) Cartoons depicting the truncation / deletion mutants of HPV-31 E1. Scale is provided in amino acid numbers of the full-length protein at the top. ( g ) Luciferase reporter assay for the antioxidant response element (ARE). Relative luciferase light units were normalized based on transfection control. The mean ± standard deviation of technical triplicates are depicted. ( h ) Model depicting how E1 influences the Keap1-Nrf2 pathway.

    Journal: Cancer discovery

    Article Title: Multiple Routes to Oncogenesis are Promoted by the Human Papillomavirus-Host Protein Network

    doi: 10.1158/2159-8290.CD-17-1018

    Figure Lengend Snippet: Functional Consequences of E1-Keap1 Interaction ( a ) Subnetwork of interaction between HPV E1 and Keap1. Blue arrows denote the main contribution of differential mutation scores to KEAP1 through network propagation. MiST score of virus-human interaction in C33A cells is indicated (0.81). The mechanism of interaction is represented by edge shapes, with +Ub indicating ubiquitination. ( b , c , d , e ) Western Blot analysis of virus-host interaction by immunoprecipitation (IP) using streptavidin-coated beads to bind the Strep-tag ( b , d , e ) or an antibody against endogenous Keap1 ( c ). Proteins indicated on the right of each blot from IP and input samples were detected using the antibodies indicated. Bands were cropped from the same original membrane. Distinction between different strains of HPV is made using strain number preceding the respective viral bait (HPV-31 = 31, HPV-16 = 16; d ). ( f ) Cartoons depicting the truncation / deletion mutants of HPV-31 E1. Scale is provided in amino acid numbers of the full-length protein at the top. ( g ) Luciferase reporter assay for the antioxidant response element (ARE). Relative luciferase light units were normalized based on transfection control. The mean ± standard deviation of technical triplicates are depicted. ( h ) Model depicting how E1 influences the Keap1-Nrf2 pathway.

    Article Snippet: Briefly, cells were seeded in 96-well plates (Costar) and transfected using PolyJet (SignaGen Laboratories) with the following plasmids: Cignal ARE Luciferase Reporter, Negative Control or Positive Control mix (25 ng), pcDNA4/TO-based pcHPV31-E1-S, pcGFP-SF or an empty vector as control (50 ng), pCDNA3.1 FLAG NRF2 (12.5 ng; Addgene #36971) and Flag-Keap1 (37.5 ng; Addgene #28023).

    Techniques: Functional Assay, Mutagenesis, Western Blot, Immunoprecipitation, Strep-tag, Luciferase, Reporter Assay, Transfection, Standard Deviation

    Keap1 knockdown sensitizes liver cells to PA-induced toxicity, and overexpression of Keap1 mutant (Keap1 ΔCTR) protects against lipotoxicity. ( a and b ) shKeap1#4 and shLuc Hep3B cells were treated with PA at 400 μ M or vehicle (V) for 6 h. ( a ) Caspase 3/7 catalytic activity was measured using a fluorogenic assay. ( b ) Cell death was determined by trypan blue exclusion assay. ( c ) Whole-cell lysates were prepared from shKeap1#4 and shLuc Hep3B cells treated with PA at 400 or 800 μ M or vehicle (V) for 6 h. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( d ) Whole-cell lysates were prepared from WT or hepatocyte-specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes. Immunoblot analysis were performed for mKeap1, mNrf2 and β -actin. ( e ) Isolated WT or Keap1 −/− HKO primary mouse hepatocytes were treated for 24 h with PA at 400 μ M or vehicle, and apoptotic nuclei were counted after DAPI staining. ( f ) Whole-cell lysates were prepared from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated at the indicated time points with PA 400 μ M or vehicle. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and β -actin. ( g ) Cell death was determined by trypan blue exclusion assay in Keap1 ΔCTR or control Hep3B cells treated with PA at 400 μ M or vehicle for 16 h. All data are expressed as mean±S.E.M. for three experiments; *P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: Keap1 knockdown sensitizes liver cells to PA-induced toxicity, and overexpression of Keap1 mutant (Keap1 ΔCTR) protects against lipotoxicity. ( a and b ) shKeap1#4 and shLuc Hep3B cells were treated with PA at 400 μ M or vehicle (V) for 6 h. ( a ) Caspase 3/7 catalytic activity was measured using a fluorogenic assay. ( b ) Cell death was determined by trypan blue exclusion assay. ( c ) Whole-cell lysates were prepared from shKeap1#4 and shLuc Hep3B cells treated with PA at 400 or 800 μ M or vehicle (V) for 6 h. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( d ) Whole-cell lysates were prepared from WT or hepatocyte-specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes. Immunoblot analysis were performed for mKeap1, mNrf2 and β -actin. ( e ) Isolated WT or Keap1 −/− HKO primary mouse hepatocytes were treated for 24 h with PA at 400 μ M or vehicle, and apoptotic nuclei were counted after DAPI staining. ( f ) Whole-cell lysates were prepared from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated at the indicated time points with PA 400 μ M or vehicle. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and β -actin. ( g ) Cell death was determined by trypan blue exclusion assay in Keap1 ΔCTR or control Hep3B cells treated with PA at 400 μ M or vehicle for 16 h. All data are expressed as mean±S.E.M. for three experiments; *P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Over Expression, Mutagenesis, Activity Assay, Trypan Blue Exclusion Assay, Knock-Out, Isolation, Staining, Stable Transfection, Transfection, Plasmid Preparation

    Keap1 knockdown induces JNK/c-Jun signaling pathway and upregulates Bim and PUMA expression. ( a–c ) Whole-cell lysates were prepared from shLuc or four shKeap1 Hep3B clones (shKeap1#1,#3, #4 and #5) ( a ) or from shLuc or shKeap1#4 Huh-7 cells ( b ) or from shLuc or shKeap1#4 HepG2 cells ( c ), and immunoblot analysis were performed for phosphorylated JNK (p-JNK), total JNK (t-JNK), phosphorylated c-Jun (p-c-Jun), c-Jun, Bim, PUMA and tubulin, a control for protein loading. ( d ) Total RNA was prepared from shLuc or shKeap1#4 Hep3B. Bim and PUMA mRNA expression were quantified by real-time PCR. Fold induction is relative to internal control GAPDH. Data represent mean±S.E.M. of three experiments; *P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: Keap1 knockdown induces JNK/c-Jun signaling pathway and upregulates Bim and PUMA expression. ( a–c ) Whole-cell lysates were prepared from shLuc or four shKeap1 Hep3B clones (shKeap1#1,#3, #4 and #5) ( a ) or from shLuc or shKeap1#4 Huh-7 cells ( b ) or from shLuc or shKeap1#4 HepG2 cells ( c ), and immunoblot analysis were performed for phosphorylated JNK (p-JNK), total JNK (t-JNK), phosphorylated c-Jun (p-c-Jun), c-Jun, Bim, PUMA and tubulin, a control for protein loading. ( d ) Total RNA was prepared from shLuc or shKeap1#4 Hep3B. Bim and PUMA mRNA expression were quantified by real-time PCR. Fold induction is relative to internal control GAPDH. Data represent mean±S.E.M. of three experiments; *P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Clone Assay, Real-time Polymerase Chain Reaction

    PA induces Keap1 protein degradation preferentially via p62-dependent autophagy. ( a ) Whole-cell lysates were prepared from Hep3B cells treated with PA (600 μ M) or vehicle (V) in the presence of the pharmacological proteasome inhibitor MG132 (5 μ M) or the pan-caspase inhibitor QVD-OPh (5 μ M) for 6 h. Immunoblot analysis were performed for Keap1 and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( b ) Whole-cell lysates were prepared from Hep3B cells treated with PA (400 μ M) for the indicated time points. Immunoblot analysis were performed for LC3-I/II and β -actin, a control for protein loading. ( c ) Hep3B cells stably expressing GFP-LC3 plasmid were treated for 4 h with PA (400 μ M). Vehicle (V)-treated cells were used as control. Next, cells were fixed with 4% paraformaldehyde, and GFP cellular expression was assessed by confocal microscopy. Nuclei were stained with DAPI. ( d and e ) Hep3B cells stably expressing shRNA targeting p62 (shp62) were treated at the indicated time points with PA at 400 μ M. Luciferase shRNA-transfected cells (shLuc) were used as control in these experiments to discount any changes to the gene expression profile that may result from the shRNA delivery method or from clonal selection. ( d ) Effective downregulation of p62 mRNA levels in shp62 cells was verified by real-time PCR. Data are expressed as mean±S.E.M. for three experiments; *P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: PA induces Keap1 protein degradation preferentially via p62-dependent autophagy. ( a ) Whole-cell lysates were prepared from Hep3B cells treated with PA (600 μ M) or vehicle (V) in the presence of the pharmacological proteasome inhibitor MG132 (5 μ M) or the pan-caspase inhibitor QVD-OPh (5 μ M) for 6 h. Immunoblot analysis were performed for Keap1 and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( b ) Whole-cell lysates were prepared from Hep3B cells treated with PA (400 μ M) for the indicated time points. Immunoblot analysis were performed for LC3-I/II and β -actin, a control for protein loading. ( c ) Hep3B cells stably expressing GFP-LC3 plasmid were treated for 4 h with PA (400 μ M). Vehicle (V)-treated cells were used as control. Next, cells were fixed with 4% paraformaldehyde, and GFP cellular expression was assessed by confocal microscopy. Nuclei were stained with DAPI. ( d and e ) Hep3B cells stably expressing shRNA targeting p62 (shp62) were treated at the indicated time points with PA at 400 μ M. Luciferase shRNA-transfected cells (shLuc) were used as control in these experiments to discount any changes to the gene expression profile that may result from the shRNA delivery method or from clonal selection. ( d ) Effective downregulation of p62 mRNA levels in shp62 cells was verified by real-time PCR. Data are expressed as mean±S.E.M. for three experiments; *P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Stable Transfection, Expressing, Plasmid Preparation, Confocal Microscopy, Staining, shRNA, Luciferase, Transfection, Selection, Real-time Polymerase Chain Reaction

    Jnk1 knockdown reduces Bim and PUMA upregulation and liver cell toxicity induced by loss of Keap1 . ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shLuc, shKeap1#4 or shKeap1#4 with shJNK1 (shKeap1#4+shJNK1), and immunoblot analysis were performed for Keap1, phosphorylated JNK (p-JNK), total JNK (t-JNK), JNK1, Bim, PUMA, PARP and β -actin. ( b ) Cell death was determined by trypan blue exclusion assay in Hep3B cells as in panel ( a ). Data are expressed as mean±S.E.M. for three experiments; * P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: Jnk1 knockdown reduces Bim and PUMA upregulation and liver cell toxicity induced by loss of Keap1 . ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shLuc, shKeap1#4 or shKeap1#4 with shJNK1 (shKeap1#4+shJNK1), and immunoblot analysis were performed for Keap1, phosphorylated JNK (p-JNK), total JNK (t-JNK), JNK1, Bim, PUMA, PARP and β -actin. ( b ) Cell death was determined by trypan blue exclusion assay in Hep3B cells as in panel ( a ). Data are expressed as mean±S.E.M. for three experiments; * P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Stable Transfection, Expressing, Trypan Blue Exclusion Assay

    Bim or PUMA knockdown reduces liver cell toxicity induced by loss of Keap1 and proposed model for PA-mediated Keap1 degradation-associated cell toxicity. ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shLuc, shKeap1#4 or shKeap1#4 with shBim (shKeap1#4+shBim) or shKeap1#4 with shPUMA (shKeap1#4+shPUMA), and immunoblot analysis were performed for Keap1, Bim, PUMA, PARP and β -actin. ( b ) Cell death was determined by trypan blue exclusion assay in Hep3B cells as in panel ( a ). Data are expressed as mean±S.E.M. for three experiments; * P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: Bim or PUMA knockdown reduces liver cell toxicity induced by loss of Keap1 and proposed model for PA-mediated Keap1 degradation-associated cell toxicity. ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shLuc, shKeap1#4 or shKeap1#4 with shBim (shKeap1#4+shBim) or shKeap1#4 with shPUMA (shKeap1#4+shPUMA), and immunoblot analysis were performed for Keap1, Bim, PUMA, PARP and β -actin. ( b ) Cell death was determined by trypan blue exclusion assay in Hep3B cells as in panel ( a ). Data are expressed as mean±S.E.M. for three experiments; * P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Stable Transfection, Expressing, Trypan Blue Exclusion Assay

    Keap1 knockdown induces spontaneous cell toxicity. ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shRNA targeting Keap1 (shKeap1). Four shRNAs (#1, #3, #4 and #5) targeting different sequences in Keap1 mRNA were used. Luciferase shRNA-transfected cells (shLuc) were used as control. Immunoblot analysis were performed for Keap1, PARP and tubulin, a control for protein loading. ( b ) Effective downregulation of Keap1 mRNA levels in shKeap1#4 cells was verified by real-time PCR. ( c ) Cell death was determined by trypan blue exclusion assay in all four shKeap1 and shLuc Hep3B clones. Data are expressed as mean±S.E.M. for three experiments; *P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: Keap1 knockdown induces spontaneous cell toxicity. ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shRNA targeting Keap1 (shKeap1). Four shRNAs (#1, #3, #4 and #5) targeting different sequences in Keap1 mRNA were used. Luciferase shRNA-transfected cells (shLuc) were used as control. Immunoblot analysis were performed for Keap1, PARP and tubulin, a control for protein loading. ( b ) Effective downregulation of Keap1 mRNA levels in shKeap1#4 cells was verified by real-time PCR. ( c ) Cell death was determined by trypan blue exclusion assay in all four shKeap1 and shLuc Hep3B clones. Data are expressed as mean±S.E.M. for three experiments; *P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Stable Transfection, Expressing, shRNA, Luciferase, Transfection, Real-time Polymerase Chain Reaction, Trypan Blue Exclusion Assay, Clone Assay

    PA-induced toxicity correlates with cellular Keap1 protein degradation and JNK activation in liver cells. ( a ) Cell death was determined by trypan blue exclusion assay in Hep3B, Huh-7 and HepG2 cells treated for 8 and 16 h with PA. The concentration of PA was 400 μ M for Hep3B and HepG2 cells and 600 μ M for Huh-7 cells. Vehicle (V)-treated cells were used as control. Data are expressed as mean±S.E.M. for three experiments; *P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: PA-induced toxicity correlates with cellular Keap1 protein degradation and JNK activation in liver cells. ( a ) Cell death was determined by trypan blue exclusion assay in Hep3B, Huh-7 and HepG2 cells treated for 8 and 16 h with PA. The concentration of PA was 400 μ M for Hep3B and HepG2 cells and 600 μ M for Huh-7 cells. Vehicle (V)-treated cells were used as control. Data are expressed as mean±S.E.M. for three experiments; *P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Activation Assay, Trypan Blue Exclusion Assay, Concentration Assay

    Cellular Keap1 protein levels regulate PA-induced JNK activation and Bim and PUMA upregulation in liver cells. ( a–e ), Whole-cell lysates were prepared from shLuc or shKeap1#4 Hep3B cells treated with PA at 400 and 800 μ M or vehicle (V) for 6 h ( a ), from shLuc or shKeap1#4 Hep3B cells treated with PA at 600 μ M at the indicated time point ( b ), from WT or hepatocyte specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes treated with PA at 600 μ M for the indicated time points ( c–d ) or from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated with PA 400 μ M at the indicated time points ( e ). Immunoblot analysis were performed for phosphorylated JNK (p-JNK), total JNK (t-JNK), Bim, PUMA, Bcl- XL and Mcl-1. Tubulin or β -actin were used as a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: Cellular Keap1 protein levels regulate PA-induced JNK activation and Bim and PUMA upregulation in liver cells. ( a–e ), Whole-cell lysates were prepared from shLuc or shKeap1#4 Hep3B cells treated with PA at 400 and 800 μ M or vehicle (V) for 6 h ( a ), from shLuc or shKeap1#4 Hep3B cells treated with PA at 600 μ M at the indicated time point ( b ), from WT or hepatocyte specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes treated with PA at 600 μ M for the indicated time points ( c–d ) or from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated with PA 400 μ M at the indicated time points ( e ). Immunoblot analysis were performed for phosphorylated JNK (p-JNK), total JNK (t-JNK), Bim, PUMA, Bcl- XL and Mcl-1. Tubulin or β -actin were used as a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Activation Assay, Knock-Out, Stable Transfection, Transfection, Mutagenesis, Plasmid Preparation

    BMSC multipotency dependent on restoration of Sox2 expression. A : Relative expression of Sox2. B : Time course of relative expression of Sox2 and YAP after Keap1 knockdown. * P

    Journal: Diabetes

    Article Title: Dysregulation of Nrf2/Keap1 Redox Pathway in Diabetes Affects Multipotency of Stromal Cells

    doi: 10.2337/db18-0232

    Figure Lengend Snippet: BMSC multipotency dependent on restoration of Sox2 expression. A : Relative expression of Sox2. B : Time course of relative expression of Sox2 and YAP after Keap1 knockdown. * P

    Article Snippet: Nrf2 (ab92946; Abcam) and Keap1 (60027-1-Ig; Proteintech) antibodies were applied and detected with fluorophore-conjugated secondary antibodies.

    Techniques: Expressing

    Model for regulation of BMSCs by the Nrf2/Keap1 pathway and disruption in diabetes. In steady state, activation of the Nrf2/Keap1 pathway preserves redox metabolism, multipotency, and associated features of BMSCs. Nrf2 drives transcription of antioxidants, metabolic modulators, and Sox2 to maintain low ROS in BMSCs and maintain multipotency. In diabetes, loss of Nrf2-driven transcription, and therefore high intracellular ROS, promotes expression of adipogenic genes and leads to commitment of BMSCs to an adipogenic fate.

    Journal: Diabetes

    Article Title: Dysregulation of Nrf2/Keap1 Redox Pathway in Diabetes Affects Multipotency of Stromal Cells

    doi: 10.2337/db18-0232

    Figure Lengend Snippet: Model for regulation of BMSCs by the Nrf2/Keap1 pathway and disruption in diabetes. In steady state, activation of the Nrf2/Keap1 pathway preserves redox metabolism, multipotency, and associated features of BMSCs. Nrf2 drives transcription of antioxidants, metabolic modulators, and Sox2 to maintain low ROS in BMSCs and maintain multipotency. In diabetes, loss of Nrf2-driven transcription, and therefore high intracellular ROS, promotes expression of adipogenic genes and leads to commitment of BMSCs to an adipogenic fate.

    Article Snippet: Nrf2 (ab92946; Abcam) and Keap1 (60027-1-Ig; Proteintech) antibodies were applied and detected with fluorophore-conjugated secondary antibodies.

    Techniques: Activation Assay, Expressing

    Nrf2/Keap1 pathway affected in altered dBMSCs. A : Detection of intracellular ROS using CM-H 2 DCFDA. ROS induces deacetylation of cell-permeant CM-H 2 DCFDA to fluorescent DCF (Ex/em 492–495/517–527 nm) that is trapped. B : Quantification of intracellular ROS. C : Comparison of ROS levels between WT-BMSCs and stromal cells using CM-H 2 DCFDA. D and E : Fluorogenic detection of ROS in cytoplasm (red) and nuclei and mitochondria (green). F : Gene expression of oxidative phosphorylation genes. G : Glycolytic indices, derived from Seahorse Mito Stress analysis. H : Mitochondrial respiratory indices, derived from Seahorse Mito Stress analysis. I : Energy map of WT and dBMSCs, comparison between P3 and P6. J and K : ELISA of nuclear Nrf2 and cytoplasmic Keap1 using lysates of WT and dBMSCs. L : Immunofluorescence of Nrf2 and Keap1 proteins in WT and dBMSCs. Magnification is 20×. M : Relative expression of Nrf2 target genes in WT vs. dBMSCs. Data represented as mean ± SD; n ≥ 3. * P . ECAR, extracellular acidification rate; OCR, oxygen consumption rate.

    Journal: Diabetes

    Article Title: Dysregulation of Nrf2/Keap1 Redox Pathway in Diabetes Affects Multipotency of Stromal Cells

    doi: 10.2337/db18-0232

    Figure Lengend Snippet: Nrf2/Keap1 pathway affected in altered dBMSCs. A : Detection of intracellular ROS using CM-H 2 DCFDA. ROS induces deacetylation of cell-permeant CM-H 2 DCFDA to fluorescent DCF (Ex/em 492–495/517–527 nm) that is trapped. B : Quantification of intracellular ROS. C : Comparison of ROS levels between WT-BMSCs and stromal cells using CM-H 2 DCFDA. D and E : Fluorogenic detection of ROS in cytoplasm (red) and nuclei and mitochondria (green). F : Gene expression of oxidative phosphorylation genes. G : Glycolytic indices, derived from Seahorse Mito Stress analysis. H : Mitochondrial respiratory indices, derived from Seahorse Mito Stress analysis. I : Energy map of WT and dBMSCs, comparison between P3 and P6. J and K : ELISA of nuclear Nrf2 and cytoplasmic Keap1 using lysates of WT and dBMSCs. L : Immunofluorescence of Nrf2 and Keap1 proteins in WT and dBMSCs. Magnification is 20×. M : Relative expression of Nrf2 target genes in WT vs. dBMSCs. Data represented as mean ± SD; n ≥ 3. * P . ECAR, extracellular acidification rate; OCR, oxygen consumption rate.

    Article Snippet: Nrf2 (ab92946; Abcam) and Keap1 (60027-1-Ig; Proteintech) antibodies were applied and detected with fluorophore-conjugated secondary antibodies.

    Techniques: Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence

    Knockdown of Keap1 restores Nrf2-mediated antioxidant and metabolic mechanisms in dBMSCs. A : Relative expression of Keap1 after silencing, 24 h posttransfection. NS served as a negative control for transfection. B : Immunoblot of nuclear and cytoplasmic lysates of untreated and silenced WT and dBMSCs, 48 h posttransfection. C and D : Relative protein expression of Nrf2 and Keap1 in cells as indicated. E : Immunofluorescence of Nrf2 and Keap1 in dBMSCs transfected with siNS or si Keap1 . Magnification is 20×. F : Flow cytometric analysis of intracellular ROS of indicated cells after Keap1 silencing, using CM-H 2 DCFDA. G : Percentage of PDGFRα + /Sca-1 + dBMSCs after si Keap1 application. H : Quantification of 8-OHdG adducts, an indicator of ROS-mediated DNA damage. I – K : Quantitative RT-PCR of Nrf2-transcribed genes as indicated. L : Total glutathione in cells. Data represented as mean ± SD; n ≥ 3. * P

    Journal: Diabetes

    Article Title: Dysregulation of Nrf2/Keap1 Redox Pathway in Diabetes Affects Multipotency of Stromal Cells

    doi: 10.2337/db18-0232

    Figure Lengend Snippet: Knockdown of Keap1 restores Nrf2-mediated antioxidant and metabolic mechanisms in dBMSCs. A : Relative expression of Keap1 after silencing, 24 h posttransfection. NS served as a negative control for transfection. B : Immunoblot of nuclear and cytoplasmic lysates of untreated and silenced WT and dBMSCs, 48 h posttransfection. C and D : Relative protein expression of Nrf2 and Keap1 in cells as indicated. E : Immunofluorescence of Nrf2 and Keap1 in dBMSCs transfected with siNS or si Keap1 . Magnification is 20×. F : Flow cytometric analysis of intracellular ROS of indicated cells after Keap1 silencing, using CM-H 2 DCFDA. G : Percentage of PDGFRα + /Sca-1 + dBMSCs after si Keap1 application. H : Quantification of 8-OHdG adducts, an indicator of ROS-mediated DNA damage. I – K : Quantitative RT-PCR of Nrf2-transcribed genes as indicated. L : Total glutathione in cells. Data represented as mean ± SD; n ≥ 3. * P

    Article Snippet: Nrf2 (ab92946; Abcam) and Keap1 (60027-1-Ig; Proteintech) antibodies were applied and detected with fluorophore-conjugated secondary antibodies.

    Techniques: Expressing, Negative Control, Transfection, Immunofluorescence, Flow Cytometry, Quantitative RT-PCR

    Nrf2 activation restores multipotency in dBMSCs. A : Stained db / db and si Keap1 -dBMSCs at P3 and 14 days in differentiation media as indicated. Panel iii magnification is 20×. B and C : Relative expression of adipogenic genes. D – F : Relative expression of osteogenic genes. G and H : Relative expression of chondrogenic genes. I : Transwell migration assay using WT or dBMSCs as indicated. J : Relative expression of cell cycle regulators. K : Proliferation in cells using MTT assay. Data represented as mean ± SD; n ≥ 3. * P

    Journal: Diabetes

    Article Title: Dysregulation of Nrf2/Keap1 Redox Pathway in Diabetes Affects Multipotency of Stromal Cells

    doi: 10.2337/db18-0232

    Figure Lengend Snippet: Nrf2 activation restores multipotency in dBMSCs. A : Stained db / db and si Keap1 -dBMSCs at P3 and 14 days in differentiation media as indicated. Panel iii magnification is 20×. B and C : Relative expression of adipogenic genes. D – F : Relative expression of osteogenic genes. G and H : Relative expression of chondrogenic genes. I : Transwell migration assay using WT or dBMSCs as indicated. J : Relative expression of cell cycle regulators. K : Proliferation in cells using MTT assay. Data represented as mean ± SD; n ≥ 3. * P

    Article Snippet: Nrf2 (ab92946; Abcam) and Keap1 (60027-1-Ig; Proteintech) antibodies were applied and detected with fluorophore-conjugated secondary antibodies.

    Techniques: Activation Assay, Staining, Expressing, Transwell Migration Assay, MTT Assay

    si Keap1 -transfected dBMSCs reconstitute role in repair. A : BMSCs from WT or db / db mice were cultured and prepared 24 h prior, with or without transfection with si Keap1 . Twenty-four hours postexcision, 5,000,000 cells in 300 μL were injected into the cutaneous wound bed and immediate periphery. The wound bed was seeded with 100 μL, and the remaining 200 μL was seeded in 50-μL aliquots in the immediate wound periphery. B : Photographs of excisional wounds inoculated with BMSCs as indicated. C : Mean time to closure of cutaneous wounds. D : Photometric quantification of wound area. E : Hematoxylin-eosin stains of tissue sections of 10-day wounds. Wound is to the left in the image. Dashed black line, wound edge epidermis. Yellow lines delineate granulation tissue area. F : CD31 immunohistochemistry on 10-day wound tissue. Scale bars, 100 μm. Data represented as mean ± SD; n = 4. ** P .

    Journal: Diabetes

    Article Title: Dysregulation of Nrf2/Keap1 Redox Pathway in Diabetes Affects Multipotency of Stromal Cells

    doi: 10.2337/db18-0232

    Figure Lengend Snippet: si Keap1 -transfected dBMSCs reconstitute role in repair. A : BMSCs from WT or db / db mice were cultured and prepared 24 h prior, with or without transfection with si Keap1 . Twenty-four hours postexcision, 5,000,000 cells in 300 μL were injected into the cutaneous wound bed and immediate periphery. The wound bed was seeded with 100 μL, and the remaining 200 μL was seeded in 50-μL aliquots in the immediate wound periphery. B : Photographs of excisional wounds inoculated with BMSCs as indicated. C : Mean time to closure of cutaneous wounds. D : Photometric quantification of wound area. E : Hematoxylin-eosin stains of tissue sections of 10-day wounds. Wound is to the left in the image. Dashed black line, wound edge epidermis. Yellow lines delineate granulation tissue area. F : CD31 immunohistochemistry on 10-day wound tissue. Scale bars, 100 μm. Data represented as mean ± SD; n = 4. ** P .

    Article Snippet: Nrf2 (ab92946; Abcam) and Keap1 (60027-1-Ig; Proteintech) antibodies were applied and detected with fluorophore-conjugated secondary antibodies.

    Techniques: Transfection, Mouse Assay, Cell Culture, Injection, Immunohistochemistry

    Keap1 ΔC competes against effects of Keap1 on the Nrf2-mediated reporter gene. ( A ) Three distinct dimers of Keap1 and Keap1 ΔC with different views were simulated by the VMD1.9.3 software, based on a template of the crystal structure of Keap1 (3WN7 deposited in PDB). ( B ) Shows schematic of the structural domains of Keap1, Keap1 ΔC , as well as other mutants Keap1 N321 and Keap1 ΔN , some of which were tagged by the V5 ectope at the N-terminal or C-terminal ends. ( C , D ) HepG2 cells were co-transfected with an Nrf2 expression construct, and both reporters of pARE-Luc and pRL-TK (as an internal control), together with Keap1, Keap1 ΔC and other mutants as indicated. After 24 h, luciferase activity was measured with significant changes ($ p

    Journal: International Journal of Molecular Sciences

    Article Title: A Naturally-Occurring Dominant-Negative Inhibitor of Keap1 Competitively against Its Negative Regulation of Nrf2

    doi: 10.3390/ijms19082150

    Figure Lengend Snippet: Keap1 ΔC competes against effects of Keap1 on the Nrf2-mediated reporter gene. ( A ) Three distinct dimers of Keap1 and Keap1 ΔC with different views were simulated by the VMD1.9.3 software, based on a template of the crystal structure of Keap1 (3WN7 deposited in PDB). ( B ) Shows schematic of the structural domains of Keap1, Keap1 ΔC , as well as other mutants Keap1 N321 and Keap1 ΔN , some of which were tagged by the V5 ectope at the N-terminal or C-terminal ends. ( C , D ) HepG2 cells were co-transfected with an Nrf2 expression construct, and both reporters of pARE-Luc and pRL-TK (as an internal control), together with Keap1, Keap1 ΔC and other mutants as indicated. After 24 h, luciferase activity was measured with significant changes ($ p

    Article Snippet: The primary antibodies such as V5 (Invitrogen, Shanghai, China), KEAP1 (D154142, Sangon Biotech, Shanghai, China), as well as Nrf2 (ab62352) and its target HO1 (ab52947), GCLM (ab126704) and NQO1 (ab80588) (all the latter four antibodies from Abcam, Shanghai, China) were herein employed.

    Techniques: Software, Transfection, Expressing, Construct, Luciferase, Activity Assay

    Keap1 ΔC has an antagonist effect on the Keap1-mediated turnover of Nrf2. ( A – D ) COS-1 cells were transfected with an Nrf2 expression plasmid alone or plus other plasmids V5-Keap1, V5-Keap1 ΔC or both, and then treated with 50 μg/mL of cycloheximide (CHX) for distinct indicated lengths of time. The total lysates were determined by Western blotting with antibodies against Nrf2, Keap1, or its V5 tag, respectively. Subsequently, the intensity of blots was quantified and shown on the bottom ( A , C ). Of note, relative abundances of Nrf2 and its stability were determined with a changing half-life within distinct setting status, which were calculated and shown graphically ( B , D ). ( E , F ) MHCC97H and ( G ) HepG2 cells were transfected with an expression construct for V5-Keap1, V5-Keap1 ΔC or empty plasmids, before being treated with 50 μg/mL of CHX for indicated times or 10 μMol/L of MG132 for 4 h, prior to being harvested in a lysis buffer. Then, distinct abundances of endogenous Nrf2 proteins were examined by immunoblotting ( E , G ). The stability of endogenous Nrf2 protein was also determined with a varying half-life within distinct setting contexts, which was shown graphically ( F ). ( H ) A model is proposed to give a clear explanation of Keap1 ΔC , acting as a dominant-negative competitor of Keap1. This is due to the fact that Keap1 ΔC can occupy the place in the formation of an invalid dimer with Keap1 or itself, no matter whether it only retains less or no ability to inhibit Nrf2. It is important to note that this mutant Keap1 ΔC has an antagonist effect on Keap1-mediated turnover of Nrf2 by proteasomal degradation pathway. In addition, upon dissociation of Nrf2 from Keap1, the CNC-bZIP factor will be allowed for spatiotemporal translocation into the nucleus before transactivating ARE-driven cytoprotective genes against oxidative stress or other biological stimuli.

    Journal: International Journal of Molecular Sciences

    Article Title: A Naturally-Occurring Dominant-Negative Inhibitor of Keap1 Competitively against Its Negative Regulation of Nrf2

    doi: 10.3390/ijms19082150

    Figure Lengend Snippet: Keap1 ΔC has an antagonist effect on the Keap1-mediated turnover of Nrf2. ( A – D ) COS-1 cells were transfected with an Nrf2 expression plasmid alone or plus other plasmids V5-Keap1, V5-Keap1 ΔC or both, and then treated with 50 μg/mL of cycloheximide (CHX) for distinct indicated lengths of time. The total lysates were determined by Western blotting with antibodies against Nrf2, Keap1, or its V5 tag, respectively. Subsequently, the intensity of blots was quantified and shown on the bottom ( A , C ). Of note, relative abundances of Nrf2 and its stability were determined with a changing half-life within distinct setting status, which were calculated and shown graphically ( B , D ). ( E , F ) MHCC97H and ( G ) HepG2 cells were transfected with an expression construct for V5-Keap1, V5-Keap1 ΔC or empty plasmids, before being treated with 50 μg/mL of CHX for indicated times or 10 μMol/L of MG132 for 4 h, prior to being harvested in a lysis buffer. Then, distinct abundances of endogenous Nrf2 proteins were examined by immunoblotting ( E , G ). The stability of endogenous Nrf2 protein was also determined with a varying half-life within distinct setting contexts, which was shown graphically ( F ). ( H ) A model is proposed to give a clear explanation of Keap1 ΔC , acting as a dominant-negative competitor of Keap1. This is due to the fact that Keap1 ΔC can occupy the place in the formation of an invalid dimer with Keap1 or itself, no matter whether it only retains less or no ability to inhibit Nrf2. It is important to note that this mutant Keap1 ΔC has an antagonist effect on Keap1-mediated turnover of Nrf2 by proteasomal degradation pathway. In addition, upon dissociation of Nrf2 from Keap1, the CNC-bZIP factor will be allowed for spatiotemporal translocation into the nucleus before transactivating ARE-driven cytoprotective genes against oxidative stress or other biological stimuli.

    Article Snippet: The primary antibodies such as V5 (Invitrogen, Shanghai, China), KEAP1 (D154142, Sangon Biotech, Shanghai, China), as well as Nrf2 (ab62352) and its target HO1 (ab52947), GCLM (ab126704) and NQO1 (ab80588) (all the latter four antibodies from Abcam, Shanghai, China) were herein employed.

    Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Construct, Lysis, Dominant Negative Mutation, Mutagenesis, Translocation Assay

    An effect of Keap1 ΔC is distinctive from Keap1 on Nrf2-target genes. ( A ) Different expression levels of endogenous proteins Keap1, Nrf2, HO-1, GCLM and NQO1 were determined by Western blotting of four distinct cell lines as indicated. β-actin served as a loading control. ( B , C ) An ARE -driven fluorescent reporter (pARE-Luc), another internal control pRL-TK, together with an expression construct for Keap1 or Keap1 ΔC , were co-transfected into MHCC97H ( B ) or HepG2 cells ( C ). After 24 h, the luciferase activity was assayed ( # p

    Journal: International Journal of Molecular Sciences

    Article Title: A Naturally-Occurring Dominant-Negative Inhibitor of Keap1 Competitively against Its Negative Regulation of Nrf2

    doi: 10.3390/ijms19082150

    Figure Lengend Snippet: An effect of Keap1 ΔC is distinctive from Keap1 on Nrf2-target genes. ( A ) Different expression levels of endogenous proteins Keap1, Nrf2, HO-1, GCLM and NQO1 were determined by Western blotting of four distinct cell lines as indicated. β-actin served as a loading control. ( B , C ) An ARE -driven fluorescent reporter (pARE-Luc), another internal control pRL-TK, together with an expression construct for Keap1 or Keap1 ΔC , were co-transfected into MHCC97H ( B ) or HepG2 cells ( C ). After 24 h, the luciferase activity was assayed ( # p

    Article Snippet: The primary antibodies such as V5 (Invitrogen, Shanghai, China), KEAP1 (D154142, Sangon Biotech, Shanghai, China), as well as Nrf2 (ab62352) and its target HO1 (ab52947), GCLM (ab126704) and NQO1 (ab80588) (all the latter four antibodies from Abcam, Shanghai, China) were herein employed.

    Techniques: Expressing, Western Blot, Construct, Transfection, Luciferase, Activity Assay

    Distinctive expression of Keap1 ΔC in different cell lines. ( A ) Two pairs of primers were designed for specific amplification of Keap1 and Keap1 ΔC . Their forward primers were identical, but their reverse primers had only four identical bases at the 3′ ends, the different colors means the difference in downstream primers. ( B ) The Keap1 - and Keap1 ΔC -specific primers were identified by almost overlapped amplification curves ( left ) and melting curves ( right ) of real-time qPCR. ( C , D ) Two distinct expression constructs for Keap1 and Keap1 ΔC were transfected into HepG2 cells. The total RNAs were extracted and subjected to real-time qPCR to detect distinctive expression of Keap1 - and Keap1 ΔC -specific mRNAs ( * p

    Journal: International Journal of Molecular Sciences

    Article Title: A Naturally-Occurring Dominant-Negative Inhibitor of Keap1 Competitively against Its Negative Regulation of Nrf2

    doi: 10.3390/ijms19082150

    Figure Lengend Snippet: Distinctive expression of Keap1 ΔC in different cell lines. ( A ) Two pairs of primers were designed for specific amplification of Keap1 and Keap1 ΔC . Their forward primers were identical, but their reverse primers had only four identical bases at the 3′ ends, the different colors means the difference in downstream primers. ( B ) The Keap1 - and Keap1 ΔC -specific primers were identified by almost overlapped amplification curves ( left ) and melting curves ( right ) of real-time qPCR. ( C , D ) Two distinct expression constructs for Keap1 and Keap1 ΔC were transfected into HepG2 cells. The total RNAs were extracted and subjected to real-time qPCR to detect distinctive expression of Keap1 - and Keap1 ΔC -specific mRNAs ( * p

    Article Snippet: The primary antibodies such as V5 (Invitrogen, Shanghai, China), KEAP1 (D154142, Sangon Biotech, Shanghai, China), as well as Nrf2 (ab62352) and its target HO1 (ab52947), GCLM (ab126704) and NQO1 (ab80588) (all the latter four antibodies from Abcam, Shanghai, China) were herein employed.

    Techniques: Expressing, Amplification, Real-time Polymerase Chain Reaction, Construct, Transfection

    A novel discovery of the Keap1 ΔC mutant. Similar but different PCR (Polymerase Chain Reaction) products of Nrf2 ( A ) and/or Keap1 ( B ) were determined by 1% Agarose gel electrophoresis of samples from six cell lines as indicated on the bottom. The red arrow and “?” indicate an unknown band. ( C ) The products of Keap1 from MHCC97H cells were identified by cDNA sequencing and then analyzed by using the Chromas software. The overlapped peaks of the sequencing curves were placed in a red box. ( D ) Single colonies of E. coli bringing a pcDNA3-based expression plasmid (in which the PCR products of Keap1 from MHCC97H cells were inserted) were subjected to identification of the inserted cDNA fragments by 1% Agarose gel electrophoresis. The black arrow indicates Keap1 and the red arrow indicates Keap1 ΔC . ( E ) A nucleotide sequence alignment of Keap1 and Keap1 ΔC was listed, using the DNAMAN software, of which the GenBack Accession No. NM_203500 sequence served as a standard. The black lines means the consensus sequence. ( F ) Shows schematic of distinct lengths of Keap1 and Keap1 ΔC mRNAs, as well as structural domains of their proteins. Abbreviations: BTB, broad complex, tramtrack and bric-à-brac domain; IVR, intervening region; DGR, double glycine repeat; and CTR, C-terminal region of Keap1.

    Journal: International Journal of Molecular Sciences

    Article Title: A Naturally-Occurring Dominant-Negative Inhibitor of Keap1 Competitively against Its Negative Regulation of Nrf2

    doi: 10.3390/ijms19082150

    Figure Lengend Snippet: A novel discovery of the Keap1 ΔC mutant. Similar but different PCR (Polymerase Chain Reaction) products of Nrf2 ( A ) and/or Keap1 ( B ) were determined by 1% Agarose gel electrophoresis of samples from six cell lines as indicated on the bottom. The red arrow and “?” indicate an unknown band. ( C ) The products of Keap1 from MHCC97H cells were identified by cDNA sequencing and then analyzed by using the Chromas software. The overlapped peaks of the sequencing curves were placed in a red box. ( D ) Single colonies of E. coli bringing a pcDNA3-based expression plasmid (in which the PCR products of Keap1 from MHCC97H cells were inserted) were subjected to identification of the inserted cDNA fragments by 1% Agarose gel electrophoresis. The black arrow indicates Keap1 and the red arrow indicates Keap1 ΔC . ( E ) A nucleotide sequence alignment of Keap1 and Keap1 ΔC was listed, using the DNAMAN software, of which the GenBack Accession No. NM_203500 sequence served as a standard. The black lines means the consensus sequence. ( F ) Shows schematic of distinct lengths of Keap1 and Keap1 ΔC mRNAs, as well as structural domains of their proteins. Abbreviations: BTB, broad complex, tramtrack and bric-à-brac domain; IVR, intervening region; DGR, double glycine repeat; and CTR, C-terminal region of Keap1.

    Article Snippet: The primary antibodies such as V5 (Invitrogen, Shanghai, China), KEAP1 (D154142, Sangon Biotech, Shanghai, China), as well as Nrf2 (ab62352) and its target HO1 (ab52947), GCLM (ab126704) and NQO1 (ab80588) (all the latter four antibodies from Abcam, Shanghai, China) were herein employed.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing, Software, Expressing, Plasmid Preparation