anti-keap1 antibody Search Results


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  • 99
    Millipore keap1 polyclonal antibody
    <t>Keap1</t> and nuclear Nrf2 expression in lung tissue after OALT. Group S: sham-operated group; Group M: normal saline control model group; Group LP: low-dose of propofol intervention group; Group HP: high-dose propofol intervention group. Data are presented as mean ± SD. (a) P
    Keap1 Polyclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti keap 1 antibody
    Effects of the iodide intake (NI, 10 HI, and 100 HI) on the changes of (a) Nrf2; (b) Keap 1; (c) Srx; and (d) Prx 3 expressions on Days 7, 14, and 28. Representative western blot and histograms of densitometric analyses normalized for the relative  β -actin content. All data is presented as the mean ± SD ( N  = 6 for each group). Statistical analyses were performed by one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test.  ∗ p
    Anti Keap 1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc anti keap1
    Gankyrin binds to the Kelch domain of <t>Keap1.</t> (A) Gankyrin influenced the binding of Keap1 to Nrf2. Equal amounts of cell lysates were immunoprecipitated with an anti-Keap1 antibody. Precipitated proteins and cell lysates were blotted with anti-Nrf2, anti-gankyrin, and anti-Keap1 antibodies. (B) Confocal microscopy was performed on HEK293T cells cotransfected with Keap1 and myc-gankyrin. Bar, 10 µm. (C and D) Gankyrin and Keap1 were cotransfected into 293T cells. Whole cell lysates were immunoprecipitated with Keap1- (C) or gankyrin-specific (D) antibodies. Precipitated proteins and cell lysates were blotted with the indicated antibodies. (E) Cell lysates from SMMC7721-con and SMMC7721-ovGank cells were immunoprecipitated with anti-Keap1 antibodies, and Western blot analysis was performed with the indicated antibodies. (F) The interaction of Myc-gankyrin with Flag-tagged truncated Keap1 fragments. The top panel shows a schematic of the truncated Keap1 fragments. HEK293T cells that were cotransfected with myc-gankyrin and Flag-tagged truncated Keap1 fragments were lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibodies. (G) The interaction of Flag-KC (Kelch domain of Keap1) with Myc-tagged gankyrin. HEK293T cells cotransfected with Flag-KC and Myc-tagged gankyrin were immunoprecipitated with anti-flag antibody and immunoblotted with anti-myc antibodies. (H) The interaction of Flag-Keap1 with Myc-tagged gankyrin mutants. The top panel shows a schematic of the gankyrin mutants. HEK293T cells cotransfected with Flag-Keap1 and myc-tagged deletion mutants of gankyrin were immunoprecipitated with anti-flag antibody. Precipitated proteins and cell lysates were blotted with anti-myc and the indicated antibodies. (I) Wild-type or ExxE motif-mutated gankyrin and Flag-Keap1 plasmids were transfected into HEK293T cells, and the cells were then lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibody. N-mutated indicated E in aa 21-24 were mutated to A, C-mutated indicated E in aa 201-204 were mutated to A, and N+C-mutated indicated E in aa 21-24 and aa 201-204 were all mutated. (J) The knockdown of Keap1 abolished the regulatory role of gankyrin on Nrf2 protein levels. Negative control oligonucleotides or siRNA targeting Keap1 were transfected into MHCCLM3-Con, -siGank, or SMMC7721-Con, -ovGank cells. Cell lysates were blotted with anti-Nrf2 and other indicated antibodies. (K) A coimmunoprecipitation assay was used to analyze the amount of gankyrin that was associated with Keap1 after stimulation with sulforaphane, tBHQ, or H 2 O 2 . SMMC7721 cells were stimulated by sulforaphane, tBHQ, or H 2 O 2 for 12 h, and the cells were then lysed and immunoprecipitated with an anti-Keap1 antibody. Precipitates and cell lysates were blotted with an anti-gankyrin antibody. The data are representative of at least two experiments with similar results.
    Anti 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
    Abcam anti keap1 antibody
    Protein levels of the <t>Keap1-Nrf2-ARE</t> pathway. (A) Keap1 protein; (B) Cytosolic and nucleic Nrf2 levels. The experiments used 18 samples from each group. Data are presented as the mean ± standard error. *P
    Anti Keap1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc keap1
    RNA sequencing reveals NRF2-activating mutation. ( a ) Schematic showing workflow for identifying an NRF2-activation gene signature. ( b ) Machine Learning Scores for sequential addition of genes identified 28 genes as the highest-scoring geneset. ( c ) Hierarchical clustering analysis of RNA sequencing data of all lung tumor (LUSC and LUAD) cases using Ward’s minimum variance method with the 28 gene signature. G1 was designated as the normal tissue cases, G2 as the NRF2-active group, and G3 as the NRF2-inactive group. Individual cases designated as normal, <t>KEAP1</t> -mutant, or NRF2 mutant are indicated with vertical lines below their clustered position. KEAP1 and NRF2 mutants were enriched in G2 relative to G3 (p
    Keap1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 642 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti keap1 antibody 1f10b6
    RNA sequencing reveals NRF2-activating mutation. ( a ) Schematic showing workflow for identifying an NRF2-activation gene signature. ( b ) Machine Learning Scores for sequential addition of genes identified 28 genes as the highest-scoring geneset. ( c ) Hierarchical clustering analysis of RNA sequencing data of all lung tumor (LUSC and LUAD) cases using Ward’s minimum variance method with the 28 gene signature. G1 was designated as the normal tissue cases, G2 as the NRF2-active group, and G3 as the NRF2-inactive group. Individual cases designated as normal, <t>KEAP1</t> -mutant, or NRF2 mutant are indicated with vertical lines below their clustered position. KEAP1 and NRF2 mutants were enriched in G2 relative to G3 (p
    Anti Keap1 Antibody 1f10b6, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Bioss polyclonal anti keap1
    RNA sequencing reveals NRF2-activating mutation. ( a ) Schematic showing workflow for identifying an NRF2-activation gene signature. ( b ) Machine Learning Scores for sequential addition of genes identified 28 genes as the highest-scoring geneset. ( c ) Hierarchical clustering analysis of RNA sequencing data of all lung tumor (LUSC and LUAD) cases using Ward’s minimum variance method with the 28 gene signature. G1 was designated as the normal tissue cases, G2 as the NRF2-active group, and G3 as the NRF2-inactive group. Individual cases designated as normal, <t>KEAP1</t> -mutant, or NRF2 mutant are indicated with vertical lines below their clustered position. KEAP1 and NRF2 mutants were enriched in G2 relative to G3 (p
    Polyclonal Anti Keap1, supplied by Bioss, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology anti keap1 antibodies
    Effects of XXT on Nrf2 and <t>Keap1</t> mRNA expression levels in HUVECs.
    Anti Keap1 Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore keap1 sigma aldrich antibodies
    Effects of XXT on Nrf2 and <t>Keap1</t> mRNA expression levels in HUVECs.
    Keap1 Sigma Aldrich Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti keap1 ab1 antibody
    Effects of XXT on Nrf2 and <t>Keap1</t> mRNA expression levels in HUVECs.
    Anti Keap1 Ab1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti keap1 antibody
    PQ regulates cell proliferation, cell death, and autophagy by modulating <t>Keap1/p65/Nrf2</t> signal pathway. a , b Western Blot was used to verify the efficiency of p65 overexpression and Nrf2 knock-down in 16HBE cells. c , d The proliferative ability of 16HBE cells treated with 500 μM of PQ for 12 h, 24 h, 48 h, 72 h, and 96 h was detected by CCK-8 assay. e , f The apoptosis ratio of 16HBE cells treated with 150 μM PQ for 2 h was detected by FCM. g – i The autophagy-associated proteins of 16HBE cells treated with 500 μM of PQ for 48 h were detected by Western Blot. Each assay had 3 repetitions (the data are presented as mean ± SD, “*” means statistical significance, p
    Monoclonal Anti Keap1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Keap1 and nuclear Nrf2 expression in lung tissue after OALT. Group S: sham-operated group; Group M: normal saline control model group; Group LP: low-dose of propofol intervention group; Group HP: high-dose propofol intervention group. Data are presented as mean ± SD. (a) P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Propofol Activation of the Nrf2 Pathway Is Associated with Amelioration of Acute Lung Injury in a Rat Liver Transplantation Model

    doi: 10.1155/2014/258567

    Figure Lengend Snippet: Keap1 and nuclear Nrf2 expression in lung tissue after OALT. Group S: sham-operated group; Group M: normal saline control model group; Group LP: low-dose of propofol intervention group; Group HP: high-dose propofol intervention group. Data are presented as mean ± SD. (a) P

    Article Snippet: Finally, the PVDF membranes were incubated with Nrf2 antibodies (1 : 250, sc-722, Santa Cruze), Keap1 antibodies (1 : 1000, ABS97, Millipore), HO-1 (1 : 250, sc-10789, Santa Cruz), or NQO1 (1 : 250, sc-32793, Santa Cruz) followed by the corresponding secondary antibodies.

    Techniques: Expressing

    A diagram of propofol-induced activation of Nrf2 under conditions of oxidative stress. Under normal conditions, a single Keap1 protein is able to target multiple Nrf2 proteins for destruction by the ubiquitin-proteasome system. Endogenously generated ROS alter the interaction between Nrf2 and its repressor under oxidative stress, resulting in the accumulation of Nrf2 in the cytoplasm and Nrf2 translocates to the nucleus. As an antioxidant, propofol enhances the effect of the endogenous activator of Nrf2 pathway. Propofol accelerates the dissociation of Nrf2 from Keap1 which leads to more Nrf2 translocation to the nucleus under conditions of oxidative stress. Through binding with Maf and ARE, Nrf2 regulates the expression of its downstream target genes (HO-1, NQO1, γ -GCS, etc.) to prevent oxidative stress and damage.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Propofol Activation of the Nrf2 Pathway Is Associated with Amelioration of Acute Lung Injury in a Rat Liver Transplantation Model

    doi: 10.1155/2014/258567

    Figure Lengend Snippet: A diagram of propofol-induced activation of Nrf2 under conditions of oxidative stress. Under normal conditions, a single Keap1 protein is able to target multiple Nrf2 proteins for destruction by the ubiquitin-proteasome system. Endogenously generated ROS alter the interaction between Nrf2 and its repressor under oxidative stress, resulting in the accumulation of Nrf2 in the cytoplasm and Nrf2 translocates to the nucleus. As an antioxidant, propofol enhances the effect of the endogenous activator of Nrf2 pathway. Propofol accelerates the dissociation of Nrf2 from Keap1 which leads to more Nrf2 translocation to the nucleus under conditions of oxidative stress. Through binding with Maf and ARE, Nrf2 regulates the expression of its downstream target genes (HO-1, NQO1, γ -GCS, etc.) to prevent oxidative stress and damage.

    Article Snippet: Finally, the PVDF membranes were incubated with Nrf2 antibodies (1 : 250, sc-722, Santa Cruze), Keap1 antibodies (1 : 1000, ABS97, Millipore), HO-1 (1 : 250, sc-10789, Santa Cruz), or NQO1 (1 : 250, sc-32793, Santa Cruz) followed by the corresponding secondary antibodies.

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

    Model for Keap1 regulation of Nrf2. Under nonoxidative conditions, Keap1 is evident in focal adhesions (FA). In addition, Keap1 and Nrf2 form a complex in the cytoplasm. This cytoplasmic location is actively maintained by Crm1/exportin, which recognizes an NES present in Keap1. In the cytoplasm, Keap1/Nrf2 is associated with the proteosome, where it is targeted for ubiquitin-mediated degradation. A small amount of the complex shuttled to the nucleus via Nrf2's NLS ensures basal levels of ARE-regulated gene transcription. Inhibition of Crm1/exportin by treatment with LMB results in nuclear translocation of both Keap1 and Nrf2. This represents influx of the cytoplasmic pool of Keap1, as the cytoskeletal pool remains in focal adhesions. Nuclear accumulation of Keap1 and Nrf2 results in the partial activation of the ARE genes. Oxidative stress modifies cysteine residues found within Keap1. This results in the release of Keap1 from the cytoskeleton and from the degradation machinery. All pools of Keap1 and Nrf2 concentrate in the nucleus. A second oxidation sensitive signal, possibly activation of PKC, which phosphorylates Nrf2 (red P), leads to full activation of ARE-regulated genes.

    Journal: Molecular and Cellular Biology

    Article Title: Keap1 Regulates the Oxidation-Sensitive Shuttling of Nrf2 into and out of the Nucleus via a Crm1-Dependent Nuclear Export Mechanism †

    doi: 10.1128/MCB.25.11.4501-4513.2005

    Figure Lengend Snippet: Model for Keap1 regulation of Nrf2. Under nonoxidative conditions, Keap1 is evident in focal adhesions (FA). In addition, Keap1 and Nrf2 form a complex in the cytoplasm. This cytoplasmic location is actively maintained by Crm1/exportin, which recognizes an NES present in Keap1. In the cytoplasm, Keap1/Nrf2 is associated with the proteosome, where it is targeted for ubiquitin-mediated degradation. A small amount of the complex shuttled to the nucleus via Nrf2's NLS ensures basal levels of ARE-regulated gene transcription. Inhibition of Crm1/exportin by treatment with LMB results in nuclear translocation of both Keap1 and Nrf2. This represents influx of the cytoplasmic pool of Keap1, as the cytoskeletal pool remains in focal adhesions. Nuclear accumulation of Keap1 and Nrf2 results in the partial activation of the ARE genes. Oxidative stress modifies cysteine residues found within Keap1. This results in the release of Keap1 from the cytoskeleton and from the degradation machinery. All pools of Keap1 and Nrf2 concentrate in the nucleus. A second oxidation sensitive signal, possibly activation of PKC, which phosphorylates Nrf2 (red P), leads to full activation of ARE-regulated genes.

    Article Snippet: For immunoblot analysis, anti-Keap1 antibody was used at a concentration of 1 μg/ml, anti-Nrf2 antibody was used at 1:1,000, mouse antiactin antibody (Sigma) was used at 1:2,500, antilamin antibody (a kind gift of the Gerace lab, TSRI, La Jolla, CA) was used at 1:1,000, rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (RDI, Flanders, NJ) was used at 0.05 μg/ml, and rabbit anti-GST alpha (Alpha Diagnostic Inc, San Antonio, TX) was used at 1:1,000.

    Techniques: Inhibition, Translocation Assay, Activation Assay

    Keap1 and Nrf2 both redistribute to the nucleus after oxidative stress. (A) Indirect immunofluorescence localization of Keap1 and Nrf2 in NIH 3T3 cells serum starved for 2 h followed by treatment for 24 h with vehicle (dimethyl sulfoxide [DMSO]) or activators of the oxidative stress pathway, DEM, tBHQ, and sulforaphane. Cells were fixed with methanol before incubation with antibodies specific to Keap1 or Nrf2 followed by incubation with an FITC-conjugated secondary antibody. For each panel, an image of the cell nuclei stained with the DNA-specific dye Hoechst (DNA) and a phase-contrast image of the cells (Phase) are also presented. (B) Localization of Keap1 in untreated HepG2 cells and cells treated for 24 h with DEM. Cells were fixed and stained as described for panel A. The corresponding immunofluorescence and phase-contrast images are shown. Bars, 10 μm. (C) Localization of Keap1 in untreated COS7 cells and cells treated with DEM for 2 h. (D) Fractionation of NIH 3T3 cells into nuclear and cytoplasmic fractions. Serum-starved NIH 3T3 cells were left untreated (−) or treated for 2 h with 100 μM DEM (+). Cells were isolated and fractionated into nuclear and cytoplasmic extracts (see Materials and Methods). Equal amounts of protein extracts were loaded in each lane, resolved by SDS-polyacrylamide gel electrophoresis, and probed for Keap1 and Nrf2 by immunoblot analysis. As loading controls, cytoplasmic extracts were probed for GAPDH and nuclear fractions were probed for Lamin A.

    Journal: Molecular and Cellular Biology

    Article Title: Keap1 Regulates the Oxidation-Sensitive Shuttling of Nrf2 into and out of the Nucleus via a Crm1-Dependent Nuclear Export Mechanism †

    doi: 10.1128/MCB.25.11.4501-4513.2005

    Figure Lengend Snippet: Keap1 and Nrf2 both redistribute to the nucleus after oxidative stress. (A) Indirect immunofluorescence localization of Keap1 and Nrf2 in NIH 3T3 cells serum starved for 2 h followed by treatment for 24 h with vehicle (dimethyl sulfoxide [DMSO]) or activators of the oxidative stress pathway, DEM, tBHQ, and sulforaphane. Cells were fixed with methanol before incubation with antibodies specific to Keap1 or Nrf2 followed by incubation with an FITC-conjugated secondary antibody. For each panel, an image of the cell nuclei stained with the DNA-specific dye Hoechst (DNA) and a phase-contrast image of the cells (Phase) are also presented. (B) Localization of Keap1 in untreated HepG2 cells and cells treated for 24 h with DEM. Cells were fixed and stained as described for panel A. The corresponding immunofluorescence and phase-contrast images are shown. Bars, 10 μm. (C) Localization of Keap1 in untreated COS7 cells and cells treated with DEM for 2 h. (D) Fractionation of NIH 3T3 cells into nuclear and cytoplasmic fractions. Serum-starved NIH 3T3 cells were left untreated (−) or treated for 2 h with 100 μM DEM (+). Cells were isolated and fractionated into nuclear and cytoplasmic extracts (see Materials and Methods). Equal amounts of protein extracts were loaded in each lane, resolved by SDS-polyacrylamide gel electrophoresis, and probed for Keap1 and Nrf2 by immunoblot analysis. As loading controls, cytoplasmic extracts were probed for GAPDH and nuclear fractions were probed for Lamin A.

    Article Snippet: For immunoblot analysis, anti-Keap1 antibody was used at a concentration of 1 μg/ml, anti-Nrf2 antibody was used at 1:1,000, mouse antiactin antibody (Sigma) was used at 1:2,500, antilamin antibody (a kind gift of the Gerace lab, TSRI, La Jolla, CA) was used at 1:1,000, rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (RDI, Flanders, NJ) was used at 0.05 μg/ml, and rabbit anti-GST alpha (Alpha Diagnostic Inc, San Antonio, TX) was used at 1:1,000.

    Techniques: Immunofluorescence, Incubation, Staining, Fractionation, Isolation, Polyacrylamide Gel Electrophoresis

    ). aa, amino acid. (B) GFP fusion constructs used to analyze the NES of Keap1. The amino acid boundaries of each construct above each diagram and the positions of the BTB, IVR, and Kelch repeat domains are shown. The position of the NES is shown with an asterisk. (C) Localization of Keap1-GFP fusion constructs in untreated NIH 3T3 cells and cells treated with 3 nM LMB for 3 h. Images were taken of live cells. Fluorescence images are arranged with the GFP fluorescence in the left panel (GFP) and the Hoechst stain of DNA in the right panel (DNA). Keap1-GFP and NES-Kelch-GFP are predominantly cytoplasmic but redistribute to the nucleus after LMB treatment. Other constructs are unaffected by LMB treatment and exhibit both nuclear and cytoplasmic locations. Bar, 10 μm. (D) A histogram quantifying the localization of the GFP constructs to the nucleus. Two hundred cells were counted for each construct. N

    Journal: Molecular and Cellular Biology

    Article Title: Keap1 Regulates the Oxidation-Sensitive Shuttling of Nrf2 into and out of the Nucleus via a Crm1-Dependent Nuclear Export Mechanism †

    doi: 10.1128/MCB.25.11.4501-4513.2005

    Figure Lengend Snippet: ). aa, amino acid. (B) GFP fusion constructs used to analyze the NES of Keap1. The amino acid boundaries of each construct above each diagram and the positions of the BTB, IVR, and Kelch repeat domains are shown. The position of the NES is shown with an asterisk. (C) Localization of Keap1-GFP fusion constructs in untreated NIH 3T3 cells and cells treated with 3 nM LMB for 3 h. Images were taken of live cells. Fluorescence images are arranged with the GFP fluorescence in the left panel (GFP) and the Hoechst stain of DNA in the right panel (DNA). Keap1-GFP and NES-Kelch-GFP are predominantly cytoplasmic but redistribute to the nucleus after LMB treatment. Other constructs are unaffected by LMB treatment and exhibit both nuclear and cytoplasmic locations. Bar, 10 μm. (D) A histogram quantifying the localization of the GFP constructs to the nucleus. Two hundred cells were counted for each construct. N

    Article Snippet: For immunoblot analysis, anti-Keap1 antibody was used at a concentration of 1 μg/ml, anti-Nrf2 antibody was used at 1:1,000, mouse antiactin antibody (Sigma) was used at 1:2,500, antilamin antibody (a kind gift of the Gerace lab, TSRI, La Jolla, CA) was used at 1:1,000, rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (RDI, Flanders, NJ) was used at 0.05 μg/ml, and rabbit anti-GST alpha (Alpha Diagnostic Inc, San Antonio, TX) was used at 1:1,000.

    Techniques: Construct, Fluorescence, Staining

    Comparison of Cys273 and Cys288 between Keap1 and human KLHL (Kelch-like) family members. Cys273 and 288 are highly conserved among mammal Keap1 but not in other KLHL family members. Note that whereas Cys273 of Keap1 corresponds to leucine (L) in other KLHL family members, Cys288 of Keap1 corresponds to glutamate (E) or glutamine (N) in other KLHL family members.

    Journal: Molecular and Cellular Biology

    Article Title: Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

    doi: 10.1128/MCB.00868-15

    Figure Lengend Snippet: Comparison of Cys273 and Cys288 between Keap1 and human KLHL (Kelch-like) family members. Cys273 and 288 are highly conserved among mammal Keap1 but not in other KLHL family members. Note that whereas Cys273 of Keap1 corresponds to leucine (L) in other KLHL family members, Cys288 of Keap1 corresponds to glutamate (E) or glutamine (N) in other KLHL family members.

    Article Snippet: Specific protein signals were detected by anti-Nrf2 , anti-HA (Roche 3F10), anti-Keap1 , or anti-α-tubulin (Sigma, DM1A) antibodies.

    Techniques:

    Keap1 C273W C288E represses Nrf2 activity in vivo . (A) A schematic presentation of KRD-Keap1 C273W C288E transgene that expresses Keap1 under the regulation of KRD is shown. (B) Growth curves for Keap1 −/− , Keap1 −/− :: Tg-Keap1 WT mice (line 34) and Keap1 −/− :: Tg-Keap1 C273W C288E mice (line 30). Note that mice of the last two genotypes grew normally. (C to F) Hematoxylin-eosin staining of esophagus transverse sections of Keap1 +/− (C), Keap1 −/− (D), Keap1 −/− :: Tg-Keap1 WT (E), and Keap1 −/− :: Tg-Keap1 C273W C288E (F) mice at P10. The arrow in panel D indicates the thickened cornified layer. (G to J) Nrf2 immunostaining of esophagus transverse sections of Keap1 +/− (G), Keap1 −/− (H), Keap1 −/− :: Tg-Keap1 WT (I), and Keap1 −/− :: Tg-Keap1 C273W C288E (J) mice at P10. Arrowheads indicate Nrf2 accumulation in basal layer cells of esophagi.

    Journal: Molecular and Cellular Biology

    Article Title: Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

    doi: 10.1128/MCB.00868-15

    Figure Lengend Snippet: Keap1 C273W C288E represses Nrf2 activity in vivo . (A) A schematic presentation of KRD-Keap1 C273W C288E transgene that expresses Keap1 under the regulation of KRD is shown. (B) Growth curves for Keap1 −/− , Keap1 −/− :: Tg-Keap1 WT mice (line 34) and Keap1 −/− :: Tg-Keap1 C273W C288E mice (line 30). Note that mice of the last two genotypes grew normally. (C to F) Hematoxylin-eosin staining of esophagus transverse sections of Keap1 +/− (C), Keap1 −/− (D), Keap1 −/− :: Tg-Keap1 WT (E), and Keap1 −/− :: Tg-Keap1 C273W C288E (F) mice at P10. The arrow in panel D indicates the thickened cornified layer. (G to J) Nrf2 immunostaining of esophagus transverse sections of Keap1 +/− (G), Keap1 −/− (H), Keap1 −/− :: Tg-Keap1 WT (I), and Keap1 −/− :: Tg-Keap1 C273W C288E (J) mice at P10. Arrowheads indicate Nrf2 accumulation in basal layer cells of esophagi.

    Article Snippet: Specific protein signals were detected by anti-Nrf2 , anti-HA (Roche 3F10), anti-Keap1 , or anti-α-tubulin (Sigma, DM1A) antibodies.

    Techniques: Activity Assay, In Vivo, Mouse Assay, Staining, Immunostaining

    Dissection of three major cysteine residues. (A) Schematic presentations of Keap1 C151S and Keap1 C273W C288E structures. (B) Keap1 C151S and Keap1 C273W C288E protein levels of stable complemented MEFs examined by Western blotting. (C to E) Keap1 WT and Keap1 C151S MEFs were treated with 0, 30, or 100 μM DEM (C), 0, 1, or 3 μM SFN (D), or 0, 10, or 30 μM tBHQ (E) for 3 h were examined by Western blotting. (F to J) Keap1 WT , Keap1 C151S , and Keap1 C273W C288E MEFs were treated with 0, 3, or 10 μM 9-OA-NO 2 (F), 0, 5, or 15 μM 4-HNE (G), 0, 3, or 10 μM NaAsO 2 (H), 0, 100, or 300 μM SNAP (I), or 0, 10, or 30 nM CDDO-Im (J) for 3 h were examined by Western blotting.

    Journal: Molecular and Cellular Biology

    Article Title: Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

    doi: 10.1128/MCB.00868-15

    Figure Lengend Snippet: Dissection of three major cysteine residues. (A) Schematic presentations of Keap1 C151S and Keap1 C273W C288E structures. (B) Keap1 C151S and Keap1 C273W C288E protein levels of stable complemented MEFs examined by Western blotting. (C to E) Keap1 WT and Keap1 C151S MEFs were treated with 0, 30, or 100 μM DEM (C), 0, 1, or 3 μM SFN (D), or 0, 10, or 30 μM tBHQ (E) for 3 h were examined by Western blotting. (F to J) Keap1 WT , Keap1 C151S , and Keap1 C273W C288E MEFs were treated with 0, 3, or 10 μM 9-OA-NO 2 (F), 0, 5, or 15 μM 4-HNE (G), 0, 3, or 10 μM NaAsO 2 (H), 0, 100, or 300 μM SNAP (I), or 0, 10, or 30 nM CDDO-Im (J) for 3 h were examined by Western blotting.

    Article Snippet: Specific protein signals were detected by anti-Nrf2 , anti-HA (Roche 3F10), anti-Keap1 , or anti-α-tubulin (Sigma, DM1A) antibodies.

    Techniques: Dissection, Western Blot

    Generation and analyses of Keap1 C288E knock-in mice. (A) Experimental scheme for isolation of thioglycolate-elicited peritoneal macrophages from wild-type and Keap1 C288E/C288E mice. Representative sequencing data show the successful replacement of a cysteine (Cys) with glutamic acid (Glu) at position 288 (C288E) of Keap1 C288E/C288E mice. (B to F) Peritoneal macrophages from WT and Keap1 C288E/C288E mice treated with 0, 3, or 10 μM 15d-PGJ 2 (B), 0, 30, or 100 μM DEM (C), 0, 3, or 10 μM 9-OA-NO 2 (D), 0, 5, or 15 μM 4-HNE (E), or 0, 10, or 30 μM PGA 2 (F) for 3 h were examined by Western blotting.

    Journal: Molecular and Cellular Biology

    Article Title: Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

    doi: 10.1128/MCB.00868-15

    Figure Lengend Snippet: Generation and analyses of Keap1 C288E knock-in mice. (A) Experimental scheme for isolation of thioglycolate-elicited peritoneal macrophages from wild-type and Keap1 C288E/C288E mice. Representative sequencing data show the successful replacement of a cysteine (Cys) with glutamic acid (Glu) at position 288 (C288E) of Keap1 C288E/C288E mice. (B to F) Peritoneal macrophages from WT and Keap1 C288E/C288E mice treated with 0, 3, or 10 μM 15d-PGJ 2 (B), 0, 30, or 100 μM DEM (C), 0, 3, or 10 μM 9-OA-NO 2 (D), 0, 5, or 15 μM 4-HNE (E), or 0, 10, or 30 μM PGA 2 (F) for 3 h were examined by Western blotting.

    Article Snippet: Specific protein signals were detected by anti-Nrf2 , anti-HA (Roche 3F10), anti-Keap1 , or anti-α-tubulin (Sigma, DM1A) antibodies.

    Techniques: Knock-In, Mouse Assay, Isolation, Sequencing, Western Blot

    Model for multiple stress-sensing mechanisms by Keap1. We examined the reactivity of three cysteine mutants against various chemical Nrf2 inducers. Based on the results, we propose a classification of Nrf2 inducers into four classes, namely, class I (Cys151 preferring), class II (Cys288 preferring), class III (Cys151/Cys273/Cys288 collaboration preferring), and class IV (Cys151/Cys273/Cys288 independent). Representative chemicals for each class are also shown in the figure. The molecular basis for the classification is of interest.

    Journal: Molecular and Cellular Biology

    Article Title: Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

    doi: 10.1128/MCB.00868-15

    Figure Lengend Snippet: Model for multiple stress-sensing mechanisms by Keap1. We examined the reactivity of three cysteine mutants against various chemical Nrf2 inducers. Based on the results, we propose a classification of Nrf2 inducers into four classes, namely, class I (Cys151 preferring), class II (Cys288 preferring), class III (Cys151/Cys273/Cys288 collaboration preferring), and class IV (Cys151/Cys273/Cys288 independent). Representative chemicals for each class are also shown in the figure. The molecular basis for the classification is of interest.

    Article Snippet: Specific protein signals were detected by anti-Nrf2 , anti-HA (Roche 3F10), anti-Keap1 , or anti-α-tubulin (Sigma, DM1A) antibodies.

    Techniques:

    Stable cell lines of Keap1 -null MEFs with Keap1 complementation. (A) Scheme for complementation of Keap1 in Keap1 −/− MEFs. PiggyBac vector expressing HA-tagged Keap1 WT cDNA and transposase expression vector were cotransfected to Keap1 −/− MEFs. Subsequently several lines of Keap1 −/− :: HA-Keap1 WT MEFs (Keap1 WT ) were established by cloning from a single colony survived after culture with puromycin. (B) Whole-cell extracts of Keap1 WT or Keap1 −/− mock cells after incubation with 0, 30, or 100 μM DEM for 3 h were examined by Western blotting. Low-, middle-, and high-level expressers of Keap1 WT MEFs are shown. (C) Schematic structure of Keap1 C273W C288E . (D) Western blot analysis of Keap1 expression in Keap1 WT and Keap1 C273W C288E MEFs. (E) Keap1 WT and Keap1 C273W C288E MEFs treated with 0, 3, or 10 μM 15d-PGJ 2 for 3 h were examined by Western blotting. (F) Graphical representation of the results shown in panel E ( n = 3). Asterisks indicate statistically significant differences (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

    doi: 10.1128/MCB.00868-15

    Figure Lengend Snippet: Stable cell lines of Keap1 -null MEFs with Keap1 complementation. (A) Scheme for complementation of Keap1 in Keap1 −/− MEFs. PiggyBac vector expressing HA-tagged Keap1 WT cDNA and transposase expression vector were cotransfected to Keap1 −/− MEFs. Subsequently several lines of Keap1 −/− :: HA-Keap1 WT MEFs (Keap1 WT ) were established by cloning from a single colony survived after culture with puromycin. (B) Whole-cell extracts of Keap1 WT or Keap1 −/− mock cells after incubation with 0, 30, or 100 μM DEM for 3 h were examined by Western blotting. Low-, middle-, and high-level expressers of Keap1 WT MEFs are shown. (C) Schematic structure of Keap1 C273W C288E . (D) Western blot analysis of Keap1 expression in Keap1 WT and Keap1 C273W C288E MEFs. (E) Keap1 WT and Keap1 C273W C288E MEFs treated with 0, 3, or 10 μM 15d-PGJ 2 for 3 h were examined by Western blotting. (F) Graphical representation of the results shown in panel E ( n = 3). Asterisks indicate statistically significant differences (*, P

    Article Snippet: Specific protein signals were detected by anti-Nrf2 , anti-HA (Roche 3F10), anti-Keap1 , or anti-α-tubulin (Sigma, DM1A) antibodies.

    Techniques: Stable Transfection, Plasmid Preparation, Expressing, Clone Assay, Incubation, Western Blot

    Novel Keap1 mutants that repress Nrf2 activity. (A) Cysteine residues of Keap1 are shown. Representative reactive cysteine residues against electrophiles are boxed. Keap1 domains: NTR (N-terminal region), BTB (broad complex, tramtrack, and bric-a-brac), IVR (intervening region), Kelch/DGR (double glycine repeat), and CTR (C-terminal region). (B and C) All 19 possible amino acid substitutions were introduced to Cys273 (B) and Cys288 (C) of human KEAP1. HEK293T cells were cotransfected with NRF2-degron LacZ (NRF2NT-LacZ) reporter plasmid (15 ng) and KEAP1 mutant expression vector (5, 15, or 45 ng) and then incubated for 48 h. The relative β-galactosidase activity was measured. Representative results from multiple independent experiments are shown. Circled and boxed terms indicate loss-of-function mutants and mutants that retain activity to repress Nrf2 accumulation, respectively. (D) HEK293T cells were cotransfected with ARE-luciferase reporter vector, Nrf2-overexpressing vector, and vector expressing 8 or 40 ng of Keap1 WT, C273S, C273W, C288S, C288E, or C273W C288E. At 24 h after transfection, the relative luciferase activity was measured. Boxes indicate mutants that retain activity to repress Nrf2 accumulation.

    Journal: Molecular and Cellular Biology

    Article Title: Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

    doi: 10.1128/MCB.00868-15

    Figure Lengend Snippet: Novel Keap1 mutants that repress Nrf2 activity. (A) Cysteine residues of Keap1 are shown. Representative reactive cysteine residues against electrophiles are boxed. Keap1 domains: NTR (N-terminal region), BTB (broad complex, tramtrack, and bric-a-brac), IVR (intervening region), Kelch/DGR (double glycine repeat), and CTR (C-terminal region). (B and C) All 19 possible amino acid substitutions were introduced to Cys273 (B) and Cys288 (C) of human KEAP1. HEK293T cells were cotransfected with NRF2-degron LacZ (NRF2NT-LacZ) reporter plasmid (15 ng) and KEAP1 mutant expression vector (5, 15, or 45 ng) and then incubated for 48 h. The relative β-galactosidase activity was measured. Representative results from multiple independent experiments are shown. Circled and boxed terms indicate loss-of-function mutants and mutants that retain activity to repress Nrf2 accumulation, respectively. (D) HEK293T cells were cotransfected with ARE-luciferase reporter vector, Nrf2-overexpressing vector, and vector expressing 8 or 40 ng of Keap1 WT, C273S, C273W, C288S, C288E, or C273W C288E. At 24 h after transfection, the relative luciferase activity was measured. Boxes indicate mutants that retain activity to repress Nrf2 accumulation.

    Article Snippet: Specific protein signals were detected by anti-Nrf2 , anti-HA (Roche 3F10), anti-Keap1 , or anti-α-tubulin (Sigma, DM1A) antibodies.

    Techniques: Activity Assay, Plasmid Preparation, Mutagenesis, Expressing, Incubation, Luciferase, Transfection

    Keap1 mutant with triple sensor cysteine mutations. (A) Schematic structure of Keap1 C151S C273W C288E . (B) Keap1 C151S C273W C288E protein levels of stable complemented MEFs examined by Western blotting. (C to L) Keap1 WT and Keap1 C151S C273W C288E MEFs treated with 0, 3, or 10 μM 9-OA-NO 2 (C), 0, 5, or 15 μM 4-HNE (D), 0, 3, or 10 μM NaAsO 2 (E), 0, 100, or 300 μM SNAP (F), 0, 10, or 30 nM CDDO-Im (G), 0, 10, or 30 μM PGA 2 (H), 0, 30, or 90 μM ZnCl 2 (I), 0, 20, or 60 μM CdCl 2 (J), 0, 3, or 10 μM Dex-Mes (K), or 0, 130, or 400 μM H 2 O 2 (L) for 3 h were examined by Western blotting.

    Journal: Molecular and Cellular Biology

    Article Title: Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

    doi: 10.1128/MCB.00868-15

    Figure Lengend Snippet: Keap1 mutant with triple sensor cysteine mutations. (A) Schematic structure of Keap1 C151S C273W C288E . (B) Keap1 C151S C273W C288E protein levels of stable complemented MEFs examined by Western blotting. (C to L) Keap1 WT and Keap1 C151S C273W C288E MEFs treated with 0, 3, or 10 μM 9-OA-NO 2 (C), 0, 5, or 15 μM 4-HNE (D), 0, 3, or 10 μM NaAsO 2 (E), 0, 100, or 300 μM SNAP (F), 0, 10, or 30 nM CDDO-Im (G), 0, 10, or 30 μM PGA 2 (H), 0, 30, or 90 μM ZnCl 2 (I), 0, 20, or 60 μM CdCl 2 (J), 0, 3, or 10 μM Dex-Mes (K), or 0, 130, or 400 μM H 2 O 2 (L) for 3 h were examined by Western blotting.

    Article Snippet: Specific protein signals were detected by anti-Nrf2 , anti-HA (Roche 3F10), anti-Keap1 , or anti-α-tubulin (Sigma, DM1A) antibodies.

    Techniques: Mutagenesis, Western Blot

    Keap1 Cys288 is a functional sensor for 15d-PGJ 2 . (A) Schematic structures of Keap1 C273W and Keap1 C288E . (B) Western blot analysis of Keap1 expression in Keap1 WT , Keap1 C273W , and Keap1 C288E MEFs. (C) Keap1 WT , Keap1 C273W , and Keap1 C288E MEFs treated with 0, 3, or 10 μM 15d-PGJ 2 for 3 h were examined by Western blotting. (D) Graphical representation of the results shown in panel C ( n = 3). Asterisks indicate statistically significant differences (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

    doi: 10.1128/MCB.00868-15

    Figure Lengend Snippet: Keap1 Cys288 is a functional sensor for 15d-PGJ 2 . (A) Schematic structures of Keap1 C273W and Keap1 C288E . (B) Western blot analysis of Keap1 expression in Keap1 WT , Keap1 C273W , and Keap1 C288E MEFs. (C) Keap1 WT , Keap1 C273W , and Keap1 C288E MEFs treated with 0, 3, or 10 μM 15d-PGJ 2 for 3 h were examined by Western blotting. (D) Graphical representation of the results shown in panel C ( n = 3). Asterisks indicate statistically significant differences (*, P

    Article Snippet: Specific protein signals were detected by anti-Nrf2 , anti-HA (Roche 3F10), anti-Keap1 , or anti-α-tubulin (Sigma, DM1A) antibodies.

    Techniques: Functional Assay, Western Blot, Expressing

    Generation and analyses of Keap1 C151S knock-in mice. (A) Experimental schema for isolation of thioglycolate-elicited peritoneal macrophages from wild-type (WT) and Keap1 C151S/C151S mice. Representative sequencing data showing replacement of a cysteine (Cys) with serine (Ser) at position 151 (C151S) of WT and Keap1 C151S/C151S mice. (B to F) Peritoneal macrophages from WT or Keap1 C151S/C151S mice were treated with 0, 30, or 100 μM DEM (B), 0, 3, or 10 μM 15d-PGJ 2 (C), 0, 10, or 30 μM PGA 2 (D), 0, 100, or 300 μM SNAP (E), or 0, 10, 30, or 100 nM CDDO-Im (F) for 3 h were examined by Western blotting.

    Journal: Molecular and Cellular Biology

    Article Title: Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

    doi: 10.1128/MCB.00868-15

    Figure Lengend Snippet: Generation and analyses of Keap1 C151S knock-in mice. (A) Experimental schema for isolation of thioglycolate-elicited peritoneal macrophages from wild-type (WT) and Keap1 C151S/C151S mice. Representative sequencing data showing replacement of a cysteine (Cys) with serine (Ser) at position 151 (C151S) of WT and Keap1 C151S/C151S mice. (B to F) Peritoneal macrophages from WT or Keap1 C151S/C151S mice were treated with 0, 30, or 100 μM DEM (B), 0, 3, or 10 μM 15d-PGJ 2 (C), 0, 10, or 30 μM PGA 2 (D), 0, 100, or 300 μM SNAP (E), or 0, 10, 30, or 100 nM CDDO-Im (F) for 3 h were examined by Western blotting.

    Article Snippet: Specific protein signals were detected by anti-Nrf2 , anti-HA (Roche 3F10), anti-Keap1 , or anti-α-tubulin (Sigma, DM1A) antibodies.

    Techniques: Knock-In, Mouse Assay, Isolation, Sequencing, Western Blot

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

    Journal: Oxidative Medicine and Cellular Longevity

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

    doi: 10.1155/2017/4383652

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

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

    Techniques: Western Blot

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

    Journal: Oxidative Medicine and Cellular Longevity

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

    doi: 10.1155/2017/4383652

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

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

    Techniques: Concentration Assay, Activation Assay

    J19-1 actives Nrf2 pathway by inhibiting Keap1-mediated Nrf2 ubiquitination in vivo in a C171 dependent manner. ( a ) HEK293 cells were transiently transfected with WT-Keap1 or C171S-Keap1, Gal4-Neh2, and HA-Ub and treated with indicated concentration of J19-1 or tBHQ for 5 h. Lysates were immunoprecipitated by anti-Gal4 antibody, and ubiquitination was assessed using anti-HA antibody. ( b ) HEK293 cells were transiently transfected with HA-Cul3 and Flag-tagged WT (or C171S) Keap1, and then treated with tBHQ or 4 μM J19-1 for 5 h. Pull-down using Flag beads and ensuing immunoblot analysis with anti-HA antibody.

    Journal: Scientific Reports

    Article Title: Pterisolic Acid B is a Nrf2 Activator by Targeting C171 within Keap1-BTB Domain

    doi: 10.1038/srep19231

    Figure Lengend Snippet: J19-1 actives Nrf2 pathway by inhibiting Keap1-mediated Nrf2 ubiquitination in vivo in a C171 dependent manner. ( a ) HEK293 cells were transiently transfected with WT-Keap1 or C171S-Keap1, Gal4-Neh2, and HA-Ub and treated with indicated concentration of J19-1 or tBHQ for 5 h. Lysates were immunoprecipitated by anti-Gal4 antibody, and ubiquitination was assessed using anti-HA antibody. ( b ) HEK293 cells were transiently transfected with HA-Cul3 and Flag-tagged WT (or C171S) Keap1, and then treated with tBHQ or 4 μM J19-1 for 5 h. Pull-down using Flag beads and ensuing immunoblot analysis with anti-HA antibody.

    Article Snippet: The antibodies of Nrf2 (ab76026), keap1 (ab66620) and Lamin A antibody (ab26300) were purchased from Abcam, the antibodies of HA, Gal4, Flag were purchased from Sigma.

    Techniques: In Vivo, Transfection, Concentration Assay, Immunoprecipitation

    J19-1 directly targets Keap1 at C171. ( a ) Domain structures of human Keap1. Locations of all cysteines and important binding partners are shown, and four key cysteines (C151, C171,C273, and C288) are highlighted in red (human Keap1). ( b ) Total lysates of Keap1-Flag transfected 293 T cells were incubated with Probe or NC at 4 °C overnight. The precipitates resolved by SDS-PAGE were stained by silver staining. ( c ) The recombinant Keap1 protein was incubated with Probe or NC at 37 °C for 1.5 h, and the mixtures were blotted for biotin or Keap1. ( d ) The recombinant Keap1 protein were incubated with Probe in the absence or presence of a 2–3 fold excess of unlabeled J19-1 for 1.5 h at 37 °C, and the mixtures were blotted for biotin or Flag. ( e ) MS/MS analysis of the recombinant Keap1 incubated with or without J19-1 for 1.5 h. The red arrow indicates the cysteine bound to J19-1 . ( f ) Recombinant wild-type (WT) Keap1 and its mutants were incubated with Probe or NC at 37 °C for 1.5 h, followed by blotting for biotin and Keap1.

    Journal: Scientific Reports

    Article Title: Pterisolic Acid B is a Nrf2 Activator by Targeting C171 within Keap1-BTB Domain

    doi: 10.1038/srep19231

    Figure Lengend Snippet: J19-1 directly targets Keap1 at C171. ( a ) Domain structures of human Keap1. Locations of all cysteines and important binding partners are shown, and four key cysteines (C151, C171,C273, and C288) are highlighted in red (human Keap1). ( b ) Total lysates of Keap1-Flag transfected 293 T cells were incubated with Probe or NC at 4 °C overnight. The precipitates resolved by SDS-PAGE were stained by silver staining. ( c ) The recombinant Keap1 protein was incubated with Probe or NC at 37 °C for 1.5 h, and the mixtures were blotted for biotin or Keap1. ( d ) The recombinant Keap1 protein were incubated with Probe in the absence or presence of a 2–3 fold excess of unlabeled J19-1 for 1.5 h at 37 °C, and the mixtures were blotted for biotin or Flag. ( e ) MS/MS analysis of the recombinant Keap1 incubated with or without J19-1 for 1.5 h. The red arrow indicates the cysteine bound to J19-1 . ( f ) Recombinant wild-type (WT) Keap1 and its mutants were incubated with Probe or NC at 37 °C for 1.5 h, followed by blotting for biotin and Keap1.

    Article Snippet: The antibodies of Nrf2 (ab76026), keap1 (ab66620) and Lamin A antibody (ab26300) were purchased from Abcam, the antibodies of HA, Gal4, Flag were purchased from Sigma.

    Techniques: Binding Assay, Transfection, Incubation, SDS Page, Staining, Silver Staining, Recombinant, Mass Spectrometry

    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

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

    Journal: Molecular Medicine Reports

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

    doi: 10.3892/mmr.2018.9288

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

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

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

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

    Journal: Molecular Medicine Reports

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

    doi: 10.3892/mmr.2018.9288

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

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

    Techniques: Expressing, Immunofluorescence, Staining, Western Blot

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

    Journal: Molecular Medicine Reports

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

    doi: 10.3892/mmr.2018.9288

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

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

    Techniques: Expressing

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

    Journal: Molecular Medicine Reports

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

    doi: 10.3892/mmr.2018.9288

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

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

    Techniques:

    Depletion and induction of NADPH quinone oxidoreductase 1 (Nqo1) changes the intracellular reactive oxygen species (ROS) levels. A, B, Relative mRNA expression (A) and protein expression (B) of nuclear factor erythroid‐derived 2‐like 2 (Nrf2)‐related antioxidants in spheroid culture of negative control (NC), Kelch‐like ECH‐associated protein 1 (Keap1), Nrf2, and Nqo1 transient knockdown (KD) cells. Keap1 KD induced Nqo1 expressions, while Nrf2 KD decreased Nqo1 expressions in spheroid culture. Protein levels are indicated below each image. C, Intracellular oxidative stress in monolayer (ML) and spheroid (SP) culture of control, Keap1, Nrf2, and Nqo1 knockdown cells. ROS levels indicated by the relative 2′,7′‐dichlorofluorescein diacetate (DCFDA) fluorescence intensity decreased in Keap1 KD cells but increased in Nrf2 or Nqo1 KD cells in spheroid culture. All data represent means ± SEM of 3 experiments. * P

    Journal: Cancer Science

    Article Title: Modulation of Nqo1 activity intercepts anoikis resistance and reduces metastatic potential of hepatocellular carcinoma, et al. Modulation of Nqo1 activity intercepts anoikis resistance and reduces metastatic potential of hepatocellular carcinoma

    doi: 10.1111/cas.14320

    Figure Lengend Snippet: Depletion and induction of NADPH quinone oxidoreductase 1 (Nqo1) changes the intracellular reactive oxygen species (ROS) levels. A, B, Relative mRNA expression (A) and protein expression (B) of nuclear factor erythroid‐derived 2‐like 2 (Nrf2)‐related antioxidants in spheroid culture of negative control (NC), Kelch‐like ECH‐associated protein 1 (Keap1), Nrf2, and Nqo1 transient knockdown (KD) cells. Keap1 KD induced Nqo1 expressions, while Nrf2 KD decreased Nqo1 expressions in spheroid culture. Protein levels are indicated below each image. C, Intracellular oxidative stress in monolayer (ML) and spheroid (SP) culture of control, Keap1, Nrf2, and Nqo1 knockdown cells. ROS levels indicated by the relative 2′,7′‐dichlorofluorescein diacetate (DCFDA) fluorescence intensity decreased in Keap1 KD cells but increased in Nrf2 or Nqo1 KD cells in spheroid culture. All data represent means ± SEM of 3 experiments. * P

    Article Snippet: Antibodies against Keap1, phosphorylated Nrf2 (Ser40), and Nqo1 were purchased from Abcam.

    Techniques: Expressing, Derivative Assay, Negative Control, Fluorescence

    Antioxidant response in anchorage‐independent culture of hepatocellular carcinoma cells. A, Examples of immunocytochemical staining of early spheroid culture (day 2). Elevated Ser40 phosphorylated nuclear factor erythroid‐derived 2‐like 2 (p‐Nrf2) and NADPH quinone oxidoreductase 1 (Nqo1) protein expressions were detected in nucleus and cytoplasm, respectively. Scale bar = 20 μm. B, C, Relative mRNA expression (B) and protein expression (C) of Kelch‐like ECH‐associated protein 1 (Keap1), Nrf2, glutathione peroxidase‐2 (GPx), and Nqo1 in monolayer (ML) and spheroid (SP) culture. Nrf2 and its target proteins, Nqo1 and GPx, were increased in spheroid culture. Protein levels are indicated below each image. D, Intracellular oxidative stress in ML and SP culture. Reactive oxygen species levels indicated by the relative 2′,7′‐dichlorofluorescein diacetate (DCFDA) fluorescence intensity increased in spheroid culture. All data represent means ± SEM of 3 experiments. * P

    Journal: Cancer Science

    Article Title: Modulation of Nqo1 activity intercepts anoikis resistance and reduces metastatic potential of hepatocellular carcinoma, et al. Modulation of Nqo1 activity intercepts anoikis resistance and reduces metastatic potential of hepatocellular carcinoma

    doi: 10.1111/cas.14320

    Figure Lengend Snippet: Antioxidant response in anchorage‐independent culture of hepatocellular carcinoma cells. A, Examples of immunocytochemical staining of early spheroid culture (day 2). Elevated Ser40 phosphorylated nuclear factor erythroid‐derived 2‐like 2 (p‐Nrf2) and NADPH quinone oxidoreductase 1 (Nqo1) protein expressions were detected in nucleus and cytoplasm, respectively. Scale bar = 20 μm. B, C, Relative mRNA expression (B) and protein expression (C) of Kelch‐like ECH‐associated protein 1 (Keap1), Nrf2, glutathione peroxidase‐2 (GPx), and Nqo1 in monolayer (ML) and spheroid (SP) culture. Nrf2 and its target proteins, Nqo1 and GPx, were increased in spheroid culture. Protein levels are indicated below each image. D, Intracellular oxidative stress in ML and SP culture. Reactive oxygen species levels indicated by the relative 2′,7′‐dichlorofluorescein diacetate (DCFDA) fluorescence intensity increased in spheroid culture. All data represent means ± SEM of 3 experiments. * P

    Article Snippet: Antibodies against Keap1, phosphorylated Nrf2 (Ser40), and Nqo1 were purchased from Abcam.

    Techniques: Staining, Derivative Assay, Expressing, Fluorescence

    Depletion and induction of NADPH quinone oxidoreductase 1 (Nqo1) alter the sensitivity of hepatocellular carcinoma cells to anoikis. A, Representative images of apoptosis/necrosis detection assay for spheroid culture on day 2 from control, Kelch‐like ECH‐associated protein 1 (Keap1), nuclear factor erythroid‐derived 2‐like 2 (Nrf2), and Nqo1 knockdown cells. Apopxin (green), 7‐AAD (red), and CytoCalcein (blue) indicate apoptosis, late apoptosis and necrosis, and viable cells, respectively. Scale bar = 200 μm. B, Graphical presentations for the proportions of viable, apoptotic, and necrotic spheroids from apoptosis/necrosis detection assays (A). Keap1 KD tended to decreased apoptosis/necrosis, whereas Nrf2 or Nqo1 KD significantly decreased viability in early detachment culture. C, Representative images of spheroids on day 7 from control, Keap1, Nrf2, and Nqo1 knockdown cells. Scale bar = 200 μm. D, Distribution of size and number of spheroids from control, Keap1, Nrf2, and Nqo1 knockdown cells. Keap1 KD significantly increased the sphere size and number of spheroids, whereas Nrf2 or Nqo1 KD significantly decreased them. Lines indicate the average values for each data points. All data represent means ± SEM of 3 experiments. * P

    Journal: Cancer Science

    Article Title: Modulation of Nqo1 activity intercepts anoikis resistance and reduces metastatic potential of hepatocellular carcinoma, et al. Modulation of Nqo1 activity intercepts anoikis resistance and reduces metastatic potential of hepatocellular carcinoma

    doi: 10.1111/cas.14320

    Figure Lengend Snippet: Depletion and induction of NADPH quinone oxidoreductase 1 (Nqo1) alter the sensitivity of hepatocellular carcinoma cells to anoikis. A, Representative images of apoptosis/necrosis detection assay for spheroid culture on day 2 from control, Kelch‐like ECH‐associated protein 1 (Keap1), nuclear factor erythroid‐derived 2‐like 2 (Nrf2), and Nqo1 knockdown cells. Apopxin (green), 7‐AAD (red), and CytoCalcein (blue) indicate apoptosis, late apoptosis and necrosis, and viable cells, respectively. Scale bar = 200 μm. B, Graphical presentations for the proportions of viable, apoptotic, and necrotic spheroids from apoptosis/necrosis detection assays (A). Keap1 KD tended to decreased apoptosis/necrosis, whereas Nrf2 or Nqo1 KD significantly decreased viability in early detachment culture. C, Representative images of spheroids on day 7 from control, Keap1, Nrf2, and Nqo1 knockdown cells. Scale bar = 200 μm. D, Distribution of size and number of spheroids from control, Keap1, Nrf2, and Nqo1 knockdown cells. Keap1 KD significantly increased the sphere size and number of spheroids, whereas Nrf2 or Nqo1 KD significantly decreased them. Lines indicate the average values for each data points. All data represent means ± SEM of 3 experiments. * P

    Article Snippet: Antibodies against Keap1, phosphorylated Nrf2 (Ser40), and Nqo1 were purchased from Abcam.

    Techniques: Detection Assay, Derivative Assay

    Cancer cells confronted with oxidative stress due to loss of scaffold and react with the nuclear factor erythroid‐derived 2‐like 2 (Nrf2)/ARE axis including NADPH quinone oxidoreductase 1 (Nqo1). Cells with anoikis resistance are able to build spheroid during circulation to the metastatic sites. β‐Lapachone (β‐Lap) futilely stimulates Nqo1 and induces excessive reactive oxygen species (ROS) to diminish the spheroid formation ability and prevent metastasis. GPx2, glutathione peroxidase‐2; Keap1, Kelch‐like ECH‐associated protein 1; Q, quinone; QH 2 , unstable hydroquinone; ARE, antioxidant response element

    Journal: Cancer Science

    Article Title: Modulation of Nqo1 activity intercepts anoikis resistance and reduces metastatic potential of hepatocellular carcinoma, et al. Modulation of Nqo1 activity intercepts anoikis resistance and reduces metastatic potential of hepatocellular carcinoma

    doi: 10.1111/cas.14320

    Figure Lengend Snippet: Cancer cells confronted with oxidative stress due to loss of scaffold and react with the nuclear factor erythroid‐derived 2‐like 2 (Nrf2)/ARE axis including NADPH quinone oxidoreductase 1 (Nqo1). Cells with anoikis resistance are able to build spheroid during circulation to the metastatic sites. β‐Lapachone (β‐Lap) futilely stimulates Nqo1 and induces excessive reactive oxygen species (ROS) to diminish the spheroid formation ability and prevent metastasis. GPx2, glutathione peroxidase‐2; Keap1, Kelch‐like ECH‐associated protein 1; Q, quinone; QH 2 , unstable hydroquinone; ARE, antioxidant response element

    Article Snippet: Antibodies against Keap1, phosphorylated Nrf2 (Ser40), and Nqo1 were purchased from Abcam.

    Techniques: Derivative Assay

    a, Site-specific chemical ubiquitylation of HA-PALB2 (1-103) at residue 20 (PALB2-K C 20-Ub) and 45 (PALB2-K C 45-Ub) was carried out by dichloroacetone linking. The resulting ubiquitylated PALB2 polypeptides along with their unmodified counterparts were subjected to pulldown with a fusion of MBP with the coiled-coil domain of BRCA1 (MBP-BRCA1-CC). I, input; PD, pulldown. Asterisk (*) indicates a non-specific band. b , Wild-type and KEAP1Δ 293T cells were treated with cycloheximide (CHX) for the indicated time and then processed for NRF2 and KEAP1 immunoblotting. Actin levels were also determined as a loading control. c , Immunoprecipitation (IP) of USP11 from extracts prepared from 293T cells that were or were not treated with camptothecin (CPT; 200 nM). IP with normal IgG was performed as a control. d , U2OS DR-GFP cells were transfected with the indicated siRNAs. 24 h post-transfection, cells were further transfected with the indicated siRNA-resistant USP11 expression vectors (WT=wild type; CS= C318S and CA= C318A catalytically-dead mutants) or an empty vector (EV), with or without an I-SceI expression vector. The percentage of GFP-positive cells was determined 48 h post-plasmid transfection for each condition and was normalized to the I-SceI + non-targeting (siCTRL) condition (mean ± s.d., N =3). e , Schematic representation of human USP11 (top) and KEAP1 (bottom) gene organization and targeting sites of sgRNAs (as described in ) used to generate the USP11Δ and USP11Δ / KEAP1Δ 293T cells. The indels introduced by the CRISPR/Cas9 and their respective frequencies are indicated. The USP11 knockout was created first and subsequently used to make the USP11Δ / KEAP1Δ double mutant. f , Immunoprecipitation (IP) of PALB2 from extracts prepared from 293T cells transfected with the indicated siRNA and with or without CPT (200 nM) treatment. IP with normal IgG was performed as a control. Extended Data Figure 1a

    Journal: Nature

    Article Title: A mechanism for the suppression of homologous recombination in G1 cells

    doi: 10.1038/nature16142

    Figure Lengend Snippet: a, Site-specific chemical ubiquitylation of HA-PALB2 (1-103) at residue 20 (PALB2-K C 20-Ub) and 45 (PALB2-K C 45-Ub) was carried out by dichloroacetone linking. The resulting ubiquitylated PALB2 polypeptides along with their unmodified counterparts were subjected to pulldown with a fusion of MBP with the coiled-coil domain of BRCA1 (MBP-BRCA1-CC). I, input; PD, pulldown. Asterisk (*) indicates a non-specific band. b , Wild-type and KEAP1Δ 293T cells were treated with cycloheximide (CHX) for the indicated time and then processed for NRF2 and KEAP1 immunoblotting. Actin levels were also determined as a loading control. c , Immunoprecipitation (IP) of USP11 from extracts prepared from 293T cells that were or were not treated with camptothecin (CPT; 200 nM). IP with normal IgG was performed as a control. d , U2OS DR-GFP cells were transfected with the indicated siRNAs. 24 h post-transfection, cells were further transfected with the indicated siRNA-resistant USP11 expression vectors (WT=wild type; CS= C318S and CA= C318A catalytically-dead mutants) or an empty vector (EV), with or without an I-SceI expression vector. The percentage of GFP-positive cells was determined 48 h post-plasmid transfection for each condition and was normalized to the I-SceI + non-targeting (siCTRL) condition (mean ± s.d., N =3). e , Schematic representation of human USP11 (top) and KEAP1 (bottom) gene organization and targeting sites of sgRNAs (as described in ) used to generate the USP11Δ and USP11Δ / KEAP1Δ 293T cells. The indels introduced by the CRISPR/Cas9 and their respective frequencies are indicated. The USP11 knockout was created first and subsequently used to make the USP11Δ / KEAP1Δ double mutant. f , Immunoprecipitation (IP) of PALB2 from extracts prepared from 293T cells transfected with the indicated siRNA and with or without CPT (200 nM) treatment. IP with normal IgG was performed as a control. Extended Data Figure 1a

    Article Snippet: We employed the following antibodies: rabbit anti-53BP1 (A300-273A, Bethyl), rabbit anti-53BP1 (sc-22760, Santa Cruz), mouse anti-53BP1 (#612523, BD Biosciences), mouse anti-γ-H2AX (clone JBW301, Millipore), rabbit anti-γ-H2AX (#2577, Cell Signaling Technologies), rabbit anti-KEAP1 (ab66620, Abcam), rabbit anti-NRF2 (ab62352, Abcam), mouse anti-Flag (clone M2, Sigma), mouse anti-tubulin (CP06, Calbiochem), mouse anti-GFP (#11814460001, Roche), mouse anti-CCNA (MONX10262, Monosan), rabbit anti-BRCA2 (ab9143, Abcam), mouse anti-BRCA2 (OP95, Calbiochem), rabbit anti-BRCA1 (#07-434, Millipore), rabbit anti-USP11 (ab109232, Abcam), rabbit anti-USP11 (A301-613A, Bethyl), rabbit anti-RAD51 (#70-001, Bioacademia), mouse anti-BrdU (RPN202, GE Healthcare), mouse anti-FK2 (BML-PW8810, Enzo), rabbit anti-PALB2 , rabbit anti-GST (sc-459, Santa Cruz), rabbit anti-CUL3 (A301-108A, Bethyl), mouse anti-MBP (E8032, NEB), mouse anti-HA (clone 12CA5, a kind gift of Dr. M. Tyers), rabbit anti-Ubiquitin (Z0458, Dako) and mouse anti-actin (CP01, Calbiochem).

    Techniques: Immunoprecipitation, Cycling Probe Technology, Transfection, Expressing, Plasmid Preparation, CRISPR, Knock-Out, Mutagenesis

    a, Quantitation of gene targeting efficiency at the LMNA locus in asynchronously dividing U2OS cells transfected with increasing amount of donor template and with (black) or without (grey) gRNAs. Gene targeting events were detected by flow cytometry (mean ± s.d., N≥ 3). b , Quantitation of gene targeting efficiency at the LMNA locus in asynchronously dividing cells transfected with the indicated siRNA. Gene targeting events were detected by flow cytometry (mean ± s.d., N =3). c , Gene targeting efficiency at the PML locus measured by flow cytometry in G1-arrested 53BP1Δ U2OS cells expressing the CtIP-T847E mutant and co-transfected with the indicated siRNA or a PALB2-KR expression construct (mean ± s.d., N =3). d , Representative FACS profiles showing the gating for 1N DNA content cells and the detection of mClover-positive cells in the LMNA gene targeting assay in asynchronous (ASN) or G1-arrested 53BP1Δ U2OS cells expressing the CtIP-T847E mutant and co-transfected with the indicated siRNA or a PALB2-KR expression construct. e , Gene targeting efficiency at the LMNA locus measured by flow cytometry in G1-arrested parental (WT) and 53BP1Δ U2OS cells transfected with KEAP1 siRNA and expressing the CtIP-T847E mutant (mean ± s.d., N =3). f , Gene targeting efficiency at the LMNA locus measured by flow cytometry in G1-arrested parental (WT) and 53BP1Δ U2OS cells transfected with the indicated siRNA and expressing either wild-type (WT) or the CtIP-T847E mutant (mean ± s.d., N =3).

    Journal: Nature

    Article Title: A mechanism for the suppression of homologous recombination in G1 cells

    doi: 10.1038/nature16142

    Figure Lengend Snippet: a, Quantitation of gene targeting efficiency at the LMNA locus in asynchronously dividing U2OS cells transfected with increasing amount of donor template and with (black) or without (grey) gRNAs. Gene targeting events were detected by flow cytometry (mean ± s.d., N≥ 3). b , Quantitation of gene targeting efficiency at the LMNA locus in asynchronously dividing cells transfected with the indicated siRNA. Gene targeting events were detected by flow cytometry (mean ± s.d., N =3). c , Gene targeting efficiency at the PML locus measured by flow cytometry in G1-arrested 53BP1Δ U2OS cells expressing the CtIP-T847E mutant and co-transfected with the indicated siRNA or a PALB2-KR expression construct (mean ± s.d., N =3). d , Representative FACS profiles showing the gating for 1N DNA content cells and the detection of mClover-positive cells in the LMNA gene targeting assay in asynchronous (ASN) or G1-arrested 53BP1Δ U2OS cells expressing the CtIP-T847E mutant and co-transfected with the indicated siRNA or a PALB2-KR expression construct. e , Gene targeting efficiency at the LMNA locus measured by flow cytometry in G1-arrested parental (WT) and 53BP1Δ U2OS cells transfected with KEAP1 siRNA and expressing the CtIP-T847E mutant (mean ± s.d., N =3). f , Gene targeting efficiency at the LMNA locus measured by flow cytometry in G1-arrested parental (WT) and 53BP1Δ U2OS cells transfected with the indicated siRNA and expressing either wild-type (WT) or the CtIP-T847E mutant (mean ± s.d., N =3).

    Article Snippet: We employed the following antibodies: rabbit anti-53BP1 (A300-273A, Bethyl), rabbit anti-53BP1 (sc-22760, Santa Cruz), mouse anti-53BP1 (#612523, BD Biosciences), mouse anti-γ-H2AX (clone JBW301, Millipore), rabbit anti-γ-H2AX (#2577, Cell Signaling Technologies), rabbit anti-KEAP1 (ab66620, Abcam), rabbit anti-NRF2 (ab62352, Abcam), mouse anti-Flag (clone M2, Sigma), mouse anti-tubulin (CP06, Calbiochem), mouse anti-GFP (#11814460001, Roche), mouse anti-CCNA (MONX10262, Monosan), rabbit anti-BRCA2 (ab9143, Abcam), mouse anti-BRCA2 (OP95, Calbiochem), rabbit anti-BRCA1 (#07-434, Millipore), rabbit anti-USP11 (ab109232, Abcam), rabbit anti-USP11 (A301-613A, Bethyl), rabbit anti-RAD51 (#70-001, Bioacademia), mouse anti-BrdU (RPN202, GE Healthcare), mouse anti-FK2 (BML-PW8810, Enzo), rabbit anti-PALB2 , rabbit anti-GST (sc-459, Santa Cruz), rabbit anti-CUL3 (A301-108A, Bethyl), mouse anti-MBP (E8032, NEB), mouse anti-HA (clone 12CA5, a kind gift of Dr. M. Tyers), rabbit anti-Ubiquitin (Z0458, Dako) and mouse anti-actin (CP01, Calbiochem).

    Techniques: Quantitation Assay, Transfection, Flow Cytometry, Cytometry, Expressing, Mutagenesis, Construct, FACS

    USP11 opposes the activity of CRL3-KEAP1 a, Normal IgG or PALB2 immunoprecipitation (IP) of extracts derived from CPT-treated 293T cells of the indicated genotypes transfected with GFP-USP11 constructs. EV, empty vector; WT, wild type; CS, C318S. b , Clonogenic survival assays of 293T cells of the indicated genotypes treated with olaparib (mean ± s.d., N≥ 3). c , Normal IgG or PALB2 IP of extracts derived from CPT-treated 293T cells of the indicated genotypes. d , Immunoblots of deubiquitylation reactions containing ubiquitylated HA-tagged PALB2 (1-103) and increasing concentrations of GST-USP11 or its C318S (CS) mutant. USP2 was used as a control. e , Cell cycle-synchronized U2OS cells were irradiated (20 Gy dose) and processed for immunoblotting. f , Immunoblots of extracts from irradiated U2OS cells transfected with the indicated siRNAs. g , Fluorescence micrographs of G1-synchronized and irradiated (20 Gy) 53BP1Δ U2OS cells transfected with the indicated siRNAs. The percentage of cells with more than 5 γ-H2AX-colocalizing BRCA2 foci is indicated (mean ± s.d., N =3).

    Journal: Nature

    Article Title: A mechanism for the suppression of homologous recombination in G1 cells

    doi: 10.1038/nature16142

    Figure Lengend Snippet: USP11 opposes the activity of CRL3-KEAP1 a, Normal IgG or PALB2 immunoprecipitation (IP) of extracts derived from CPT-treated 293T cells of the indicated genotypes transfected with GFP-USP11 constructs. EV, empty vector; WT, wild type; CS, C318S. b , Clonogenic survival assays of 293T cells of the indicated genotypes treated with olaparib (mean ± s.d., N≥ 3). c , Normal IgG or PALB2 IP of extracts derived from CPT-treated 293T cells of the indicated genotypes. d , Immunoblots of deubiquitylation reactions containing ubiquitylated HA-tagged PALB2 (1-103) and increasing concentrations of GST-USP11 or its C318S (CS) mutant. USP2 was used as a control. e , Cell cycle-synchronized U2OS cells were irradiated (20 Gy dose) and processed for immunoblotting. f , Immunoblots of extracts from irradiated U2OS cells transfected with the indicated siRNAs. g , Fluorescence micrographs of G1-synchronized and irradiated (20 Gy) 53BP1Δ U2OS cells transfected with the indicated siRNAs. The percentage of cells with more than 5 γ-H2AX-colocalizing BRCA2 foci is indicated (mean ± s.d., N =3).

    Article Snippet: We employed the following antibodies: rabbit anti-53BP1 (A300-273A, Bethyl), rabbit anti-53BP1 (sc-22760, Santa Cruz), mouse anti-53BP1 (#612523, BD Biosciences), mouse anti-γ-H2AX (clone JBW301, Millipore), rabbit anti-γ-H2AX (#2577, Cell Signaling Technologies), rabbit anti-KEAP1 (ab66620, Abcam), rabbit anti-NRF2 (ab62352, Abcam), mouse anti-Flag (clone M2, Sigma), mouse anti-tubulin (CP06, Calbiochem), mouse anti-GFP (#11814460001, Roche), mouse anti-CCNA (MONX10262, Monosan), rabbit anti-BRCA2 (ab9143, Abcam), mouse anti-BRCA2 (OP95, Calbiochem), rabbit anti-BRCA1 (#07-434, Millipore), rabbit anti-USP11 (ab109232, Abcam), rabbit anti-USP11 (A301-613A, Bethyl), rabbit anti-RAD51 (#70-001, Bioacademia), mouse anti-BrdU (RPN202, GE Healthcare), mouse anti-FK2 (BML-PW8810, Enzo), rabbit anti-PALB2 , rabbit anti-GST (sc-459, Santa Cruz), rabbit anti-CUL3 (A301-108A, Bethyl), mouse anti-MBP (E8032, NEB), mouse anti-HA (clone 12CA5, a kind gift of Dr. M. Tyers), rabbit anti-Ubiquitin (Z0458, Dako) and mouse anti-actin (CP01, Calbiochem).

    Techniques: Activity Assay, Immunoprecipitation, Derivative Assay, Cycling Probe Technology, Transfection, Construct, Plasmid Preparation, Western Blot, Mutagenesis, Irradiation, Fluorescence

    Ubiquitylation of PALB2 prevents BRCA1-PALB2 interaction a, Sequence of the PALB2 N-terminus and mutants. b , GFP IP of extracts derived from G1- or S-phase synchronized 293T cells expressing the indicated GFP-PALB2 proteins. c , In vitro ubiquitylation of the indicated HA-tagged PALB2 proteins by CRL3-KEAP1. d , Pulldown assay of ubiquitylated HA-PALB2 (1-103) incubated with MBP or MBP-BRCA1-CC. I: input, PD: pulldown, FT: flow-through. The asterisk denotes a fragment of HA-PALB2 competent for BRCA1 binding.

    Journal: Nature

    Article Title: A mechanism for the suppression of homologous recombination in G1 cells

    doi: 10.1038/nature16142

    Figure Lengend Snippet: Ubiquitylation of PALB2 prevents BRCA1-PALB2 interaction a, Sequence of the PALB2 N-terminus and mutants. b , GFP IP of extracts derived from G1- or S-phase synchronized 293T cells expressing the indicated GFP-PALB2 proteins. c , In vitro ubiquitylation of the indicated HA-tagged PALB2 proteins by CRL3-KEAP1. d , Pulldown assay of ubiquitylated HA-PALB2 (1-103) incubated with MBP or MBP-BRCA1-CC. I: input, PD: pulldown, FT: flow-through. The asterisk denotes a fragment of HA-PALB2 competent for BRCA1 binding.

    Article Snippet: We employed the following antibodies: rabbit anti-53BP1 (A300-273A, Bethyl), rabbit anti-53BP1 (sc-22760, Santa Cruz), mouse anti-53BP1 (#612523, BD Biosciences), mouse anti-γ-H2AX (clone JBW301, Millipore), rabbit anti-γ-H2AX (#2577, Cell Signaling Technologies), rabbit anti-KEAP1 (ab66620, Abcam), rabbit anti-NRF2 (ab62352, Abcam), mouse anti-Flag (clone M2, Sigma), mouse anti-tubulin (CP06, Calbiochem), mouse anti-GFP (#11814460001, Roche), mouse anti-CCNA (MONX10262, Monosan), rabbit anti-BRCA2 (ab9143, Abcam), mouse anti-BRCA2 (OP95, Calbiochem), rabbit anti-BRCA1 (#07-434, Millipore), rabbit anti-USP11 (ab109232, Abcam), rabbit anti-USP11 (A301-613A, Bethyl), rabbit anti-RAD51 (#70-001, Bioacademia), mouse anti-BrdU (RPN202, GE Healthcare), mouse anti-FK2 (BML-PW8810, Enzo), rabbit anti-PALB2 , rabbit anti-GST (sc-459, Santa Cruz), rabbit anti-CUL3 (A301-108A, Bethyl), mouse anti-MBP (E8032, NEB), mouse anti-HA (clone 12CA5, a kind gift of Dr. M. Tyers), rabbit anti-Ubiquitin (Z0458, Dako) and mouse anti-actin (CP01, Calbiochem).

    Techniques: Sequencing, Derivative Assay, Expressing, In Vitro, Incubation, Flow Cytometry, Binding Assay

    Reactivation of HR in G1 a, Quantitation of wild type (WT) and 53BP1Δ U2OS cells co-transfected with non-targeting (CTRL) or KEAP1 siRNAs and vectors expressing WT CtIP or the T847E (TE) mutant that were synchronized in G1, irradiated (2 Gy) and processed for γ-H2AX and RAD51 immunofluorescence (mean ± s.d., N =3). b , Representative micrographs from a. c , Schematic of the gene targeting assay. d , Gene targeting efficiency at the LMNA locus in asynchronously dividing (ASN) and G1-arrested U2OS cells (mean ± s.d., N =3). e , Gene targeting at the LMNA locus in G1-arrested cells transfected with the indicated siRNA or a PALB2-KR expression vector (mean ± s.d., N =3). f , Model of the cell-cycle regulation of HR.

    Journal: Nature

    Article Title: A mechanism for the suppression of homologous recombination in G1 cells

    doi: 10.1038/nature16142

    Figure Lengend Snippet: Reactivation of HR in G1 a, Quantitation of wild type (WT) and 53BP1Δ U2OS cells co-transfected with non-targeting (CTRL) or KEAP1 siRNAs and vectors expressing WT CtIP or the T847E (TE) mutant that were synchronized in G1, irradiated (2 Gy) and processed for γ-H2AX and RAD51 immunofluorescence (mean ± s.d., N =3). b , Representative micrographs from a. c , Schematic of the gene targeting assay. d , Gene targeting efficiency at the LMNA locus in asynchronously dividing (ASN) and G1-arrested U2OS cells (mean ± s.d., N =3). e , Gene targeting at the LMNA locus in G1-arrested cells transfected with the indicated siRNA or a PALB2-KR expression vector (mean ± s.d., N =3). f , Model of the cell-cycle regulation of HR.

    Article Snippet: We employed the following antibodies: rabbit anti-53BP1 (A300-273A, Bethyl), rabbit anti-53BP1 (sc-22760, Santa Cruz), mouse anti-53BP1 (#612523, BD Biosciences), mouse anti-γ-H2AX (clone JBW301, Millipore), rabbit anti-γ-H2AX (#2577, Cell Signaling Technologies), rabbit anti-KEAP1 (ab66620, Abcam), rabbit anti-NRF2 (ab62352, Abcam), mouse anti-Flag (clone M2, Sigma), mouse anti-tubulin (CP06, Calbiochem), mouse anti-GFP (#11814460001, Roche), mouse anti-CCNA (MONX10262, Monosan), rabbit anti-BRCA2 (ab9143, Abcam), mouse anti-BRCA2 (OP95, Calbiochem), rabbit anti-BRCA1 (#07-434, Millipore), rabbit anti-USP11 (ab109232, Abcam), rabbit anti-USP11 (A301-613A, Bethyl), rabbit anti-RAD51 (#70-001, Bioacademia), mouse anti-BrdU (RPN202, GE Healthcare), mouse anti-FK2 (BML-PW8810, Enzo), rabbit anti-PALB2 , rabbit anti-GST (sc-459, Santa Cruz), rabbit anti-CUL3 (A301-108A, Bethyl), mouse anti-MBP (E8032, NEB), mouse anti-HA (clone 12CA5, a kind gift of Dr. M. Tyers), rabbit anti-Ubiquitin (Z0458, Dako) and mouse anti-actin (CP01, Calbiochem).

    Techniques: Quantitation Assay, Transfection, Expressing, Mutagenesis, Irradiation, Immunofluorescence, Plasmid Preparation

    a, Representative micrographs of the experiment shown in . b , Schematic representation of human KEAP1 . a. The indels introduced by CRISPR/Cas9 and their respective frequencies are indicated. c , Immunoprecipitation (IP) of PALB2 from extracts prepared from irradiated 293T cells. IP with normal IgG was performed as a control. d , 293T cells with the indicated genotypes were transfected with the indicated HA-KEAP1 constructs, synchronized in G1 or S phases and irradiated. Cells were processed for PALB2 immunoprecipitation (IP). EV, empty vector. e , Quantification of U2OS 256 cells transfected with the indicated GFP-PALB2 mutants and mCherry-LacR-BRCA1. Cells were also stained with a Cyclin A antibody to determine cell cycle position ( N =3). f , Quantification of U2OS 256 cells transfected with GFP-PALB2 and mCherry-LacR-BRCA1-CC (WT or K1406R mutant). Cells were also stained with a Cyclin A antibody to determine cell cycle position. This panel shows that the sole lysine in the PALB2-interaction motif of BRCA1 is not involved in the cell cycle regulation of the PALB2-BRCA1 interaction. Fig. 1d

    Journal: Nature

    Article Title: A mechanism for the suppression of homologous recombination in G1 cells

    doi: 10.1038/nature16142

    Figure Lengend Snippet: a, Representative micrographs of the experiment shown in . b , Schematic representation of human KEAP1 . a. The indels introduced by CRISPR/Cas9 and their respective frequencies are indicated. c , Immunoprecipitation (IP) of PALB2 from extracts prepared from irradiated 293T cells. IP with normal IgG was performed as a control. d , 293T cells with the indicated genotypes were transfected with the indicated HA-KEAP1 constructs, synchronized in G1 or S phases and irradiated. Cells were processed for PALB2 immunoprecipitation (IP). EV, empty vector. e , Quantification of U2OS 256 cells transfected with the indicated GFP-PALB2 mutants and mCherry-LacR-BRCA1. Cells were also stained with a Cyclin A antibody to determine cell cycle position ( N =3). f , Quantification of U2OS 256 cells transfected with GFP-PALB2 and mCherry-LacR-BRCA1-CC (WT or K1406R mutant). Cells were also stained with a Cyclin A antibody to determine cell cycle position. This panel shows that the sole lysine in the PALB2-interaction motif of BRCA1 is not involved in the cell cycle regulation of the PALB2-BRCA1 interaction. Fig. 1d

    Article Snippet: We employed the following antibodies: rabbit anti-53BP1 (A300-273A, Bethyl), rabbit anti-53BP1 (sc-22760, Santa Cruz), mouse anti-53BP1 (#612523, BD Biosciences), mouse anti-γ-H2AX (clone JBW301, Millipore), rabbit anti-γ-H2AX (#2577, Cell Signaling Technologies), rabbit anti-KEAP1 (ab66620, Abcam), rabbit anti-NRF2 (ab62352, Abcam), mouse anti-Flag (clone M2, Sigma), mouse anti-tubulin (CP06, Calbiochem), mouse anti-GFP (#11814460001, Roche), mouse anti-CCNA (MONX10262, Monosan), rabbit anti-BRCA2 (ab9143, Abcam), mouse anti-BRCA2 (OP95, Calbiochem), rabbit anti-BRCA1 (#07-434, Millipore), rabbit anti-USP11 (ab109232, Abcam), rabbit anti-USP11 (A301-613A, Bethyl), rabbit anti-RAD51 (#70-001, Bioacademia), mouse anti-BrdU (RPN202, GE Healthcare), mouse anti-FK2 (BML-PW8810, Enzo), rabbit anti-PALB2 , rabbit anti-GST (sc-459, Santa Cruz), rabbit anti-CUL3 (A301-108A, Bethyl), mouse anti-MBP (E8032, NEB), mouse anti-HA (clone 12CA5, a kind gift of Dr. M. Tyers), rabbit anti-Ubiquitin (Z0458, Dako) and mouse anti-actin (CP01, Calbiochem).

    Techniques: CRISPR, Immunoprecipitation, Irradiation, Transfection, Construct, Plasmid Preparation, Staining, Mutagenesis

    RA enhances the effects of TMZ on Keap1/Nrf2/ARE signaling. (A) Keap1/Nrf2/ARE mRNA levels detected by RT-PCR. (B) Protein levels were detected using western blot analysis. Bar charts show relative protein levels in the treatment and control groups. Statistically significant difference (*P

    Journal: Oncology Letters

    Article Title: All-trans retinoic acid enhances temozolomide-induced autophagy in human glioma cells U251 via targeting Keap1/Nrf2/ARE signaling pathway

    doi: 10.3892/ol.2017.6482

    Figure Lengend Snippet: RA enhances the effects of TMZ on Keap1/Nrf2/ARE signaling. (A) Keap1/Nrf2/ARE mRNA levels detected by RT-PCR. (B) Protein levels were detected using western blot analysis. Bar charts show relative protein levels in the treatment and control groups. Statistically significant difference (*P

    Article Snippet: After 1 h of blocking with the 5% non-fat milk, the membranes were incubated with the primary rabbit anti-LC3B, the rabbit anti-Beclin 1 antibody, the rabbit anti-Keap1 antibody, the rabbit anti-Nrf2 antibody, the rabbit anti-ARE antibody (1:1,000; Abcam, Cambridge, MA, USA) at 4°C overnight.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

    Changes in the Nrf2 and NF-κB signaling pathways in HaCaT cells after UV irradiation. (A) Nuclear and cytoplasmic proteins were extracted from the cultured cells at 0, 0.5, 1, 2, 3, 4, 5 and 6 h after UV irradiation (90 mJ/cm 2 ). The protein expression levels of NF-κB-mediated p65, and IκBα and Nrf2-mediated Nrf2 and keap1, were measured by western blotting. (B) Relative changes in protein intensities were quantified by densitometric analysis and are presented as bar diagrams (n=3 for each group). The results are expressed as the mean ± SD. a P

    Journal: International Journal of Molecular Medicine

    Article Title: Andrographolide sodium bisulfate attenuates UV-induced photo-damage by activating the keap1/Nrf2 pathway and downregulating the NF-κB pathway in HaCaT keratinocytes

    doi: 10.3892/ijmm.2019.4415

    Figure Lengend Snippet: Changes in the Nrf2 and NF-κB signaling pathways in HaCaT cells after UV irradiation. (A) Nuclear and cytoplasmic proteins were extracted from the cultured cells at 0, 0.5, 1, 2, 3, 4, 5 and 6 h after UV irradiation (90 mJ/cm 2 ). The protein expression levels of NF-κB-mediated p65, and IκBα and Nrf2-mediated Nrf2 and keap1, were measured by western blotting. (B) Relative changes in protein intensities were quantified by densitometric analysis and are presented as bar diagrams (n=3 for each group). The results are expressed as the mean ± SD. a P

    Article Snippet: Anti-Nrf2 rabbit antibody (cat. no. ab62352), anti-keap1 rabbit antibody (cat. no. ab218815), anti-IκBα rabbit antibody (cat. no. ab32518) and anti-Lamin B1 rabbit antibody (cat. no. ab16048) were purchased from Abcam.

    Techniques: Irradiation, Cell Culture, Expressing, Western Blot

    ASB activates the Nrf2 signaling pathway in UV-induced HaCaT cells. The cells were preincubated with ASB (10, 30 and 100 µ M) and irradiated with UV (90 mJ/cm 2 ). (A) Nuclear and cytoplasmic proteins were extracted; Nrf2 and keap1 proteins were measured by western blotting. Relative changes in protein intensity were quantified for (B) keap1, (C) cytosolic Nrf2 and (D) nuclear Nrf2 by densitometric analysis, and are presented as bar diagrams (n=3 for each group). # P

    Journal: International Journal of Molecular Medicine

    Article Title: Andrographolide sodium bisulfate attenuates UV-induced photo-damage by activating the keap1/Nrf2 pathway and downregulating the NF-κB pathway in HaCaT keratinocytes

    doi: 10.3892/ijmm.2019.4415

    Figure Lengend Snippet: ASB activates the Nrf2 signaling pathway in UV-induced HaCaT cells. The cells were preincubated with ASB (10, 30 and 100 µ M) and irradiated with UV (90 mJ/cm 2 ). (A) Nuclear and cytoplasmic proteins were extracted; Nrf2 and keap1 proteins were measured by western blotting. Relative changes in protein intensity were quantified for (B) keap1, (C) cytosolic Nrf2 and (D) nuclear Nrf2 by densitometric analysis, and are presented as bar diagrams (n=3 for each group). # P

    Article Snippet: Anti-Nrf2 rabbit antibody (cat. no. ab62352), anti-keap1 rabbit antibody (cat. no. ab218815), anti-IκBα rabbit antibody (cat. no. ab32518) and anti-Lamin B1 rabbit antibody (cat. no. ab16048) were purchased from Abcam.

    Techniques: Irradiation, Western Blot

    Schematic diagram of the mechanism of action of ASB in UV-induced photo-damage in HaCaT cells. UV, ultraviolet; ASB, androgra-pholide sodium bisulfite; ROS, reactive oxygen species; keap1, kelch-like ECH-associated protein 1; Nrf2, nuclear factor E2-related factor 2; IL, interleukin; TNF-α, tumor necrosis factor-α; GCLC, glutamate-cysteine ligase catalytic subunit; NQO1, NAD(P)H quinone oxidoreductase 1; IκBα, NF-κB inhibitor-α; ARE, antioxidant response element.

    Journal: International Journal of Molecular Medicine

    Article Title: Andrographolide sodium bisulfate attenuates UV-induced photo-damage by activating the keap1/Nrf2 pathway and downregulating the NF-κB pathway in HaCaT keratinocytes

    doi: 10.3892/ijmm.2019.4415

    Figure Lengend Snippet: Schematic diagram of the mechanism of action of ASB in UV-induced photo-damage in HaCaT cells. UV, ultraviolet; ASB, androgra-pholide sodium bisulfite; ROS, reactive oxygen species; keap1, kelch-like ECH-associated protein 1; Nrf2, nuclear factor E2-related factor 2; IL, interleukin; TNF-α, tumor necrosis factor-α; GCLC, glutamate-cysteine ligase catalytic subunit; NQO1, NAD(P)H quinone oxidoreductase 1; IκBα, NF-κB inhibitor-α; ARE, antioxidant response element.

    Article Snippet: Anti-Nrf2 rabbit antibody (cat. no. ab62352), anti-keap1 rabbit antibody (cat. no. ab218815), anti-IκBα rabbit antibody (cat. no. ab32518) and anti-Lamin B1 rabbit antibody (cat. no. ab16048) were purchased from Abcam.

    Techniques:

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

    Journal: The Journal of Experimental Medicine

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

    doi: 10.1084/jem.20151208

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

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

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

    Protein levels of the Keap1-Nrf2-ARE pathway. (A) Keap1 protein; (B) Cytosolic and nucleic Nrf2 levels. The experiments used 18 samples from each group. Data are presented as the mean ± standard error. *P

    Journal: Molecular Medicine Reports

    Article Title: Curcumin alleviates liver oxidative stress in type 1 diabetic rats

    doi: 10.3892/mmr.2017.7911

    Figure Lengend Snippet: Protein levels of the Keap1-Nrf2-ARE pathway. (A) Keap1 protein; (B) Cytosolic and nucleic Nrf2 levels. The experiments used 18 samples from each group. Data are presented as the mean ± standard error. *P

    Article Snippet: After blocking, the protein was incubated overnight at 4°C with anti-keap1 antibody (1:1,500, Abcam) and anti-Nrf2 antibody (1:1,000, Abcam).

    Techniques:

    Diagram illustrating the proposed mechanism of action of curcumin. Keap1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor (erythroid-derived 2)-like 2; HO-1, heme oxygenase-1; NQO-1, norvegicus NAD(P)H quinone dehydrogenase 1; GSH-Px, glutathione peroxidase; CAT, catalase; SOD1, superoxide dismutase 1; Streptozotocin; OS, oxidative stress.

    Journal: Molecular Medicine Reports

    Article Title: Curcumin alleviates liver oxidative stress in type 1 diabetic rats

    doi: 10.3892/mmr.2017.7911

    Figure Lengend Snippet: Diagram illustrating the proposed mechanism of action of curcumin. Keap1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor (erythroid-derived 2)-like 2; HO-1, heme oxygenase-1; NQO-1, norvegicus NAD(P)H quinone dehydrogenase 1; GSH-Px, glutathione peroxidase; CAT, catalase; SOD1, superoxide dismutase 1; Streptozotocin; OS, oxidative stress.

    Article Snippet: After blocking, the protein was incubated overnight at 4°C with anti-keap1 antibody (1:1,500, Abcam) and anti-Nrf2 antibody (1:1,000, Abcam).

    Techniques: Derivative Assay

    RNA sequencing reveals NRF2-activating mutation. ( a ) Schematic showing workflow for identifying an NRF2-activation gene signature. ( b ) Machine Learning Scores for sequential addition of genes identified 28 genes as the highest-scoring geneset. ( c ) Hierarchical clustering analysis of RNA sequencing data of all lung tumor (LUSC and LUAD) cases using Ward’s minimum variance method with the 28 gene signature. G1 was designated as the normal tissue cases, G2 as the NRF2-active group, and G3 as the NRF2-inactive group. Individual cases designated as normal, KEAP1 -mutant, or NRF2 mutant are indicated with vertical lines below their clustered position. KEAP1 and NRF2 mutants were enriched in G2 relative to G3 (p

    Journal: Scientific Reports

    Article Title: A catalogue of somatic NRF2 gain-of-function mutations in cancer

    doi: 10.1038/s41598-018-31281-0

    Figure Lengend Snippet: RNA sequencing reveals NRF2-activating mutation. ( a ) Schematic showing workflow for identifying an NRF2-activation gene signature. ( b ) Machine Learning Scores for sequential addition of genes identified 28 genes as the highest-scoring geneset. ( c ) Hierarchical clustering analysis of RNA sequencing data of all lung tumor (LUSC and LUAD) cases using Ward’s minimum variance method with the 28 gene signature. G1 was designated as the normal tissue cases, G2 as the NRF2-active group, and G3 as the NRF2-inactive group. Individual cases designated as normal, KEAP1 -mutant, or NRF2 mutant are indicated with vertical lines below their clustered position. KEAP1 and NRF2 mutants were enriched in G2 relative to G3 (p

    Article Snippet: Primary antibodies used in this study were raised against β-actin (ACTB) (1:10,000 in milk, Sigma A1978, St. Louis, MO), HA (1:1000 in milk, Cell Signaling, Danvers, MA) MYC (1:1000 in milk, Cell Signaling 2276), and KEAP1 (1:1000 in milk, Cell Signaling 4678).

    Techniques: RNA Sequencing Assay, Mutagenesis, Activation Assay

    NRF2 and KEAP1 mutations are overrepresented in tumors associated with carcinogen exposure. ( a ) Number of cases identified with NRF2 mutation, KEAP1 mutation, or both. ( b ) Percentage of cases within tumor types harboring non-synonymous NRF2 mutations. ( c ) Percentage of cases within tumor types harboring non-synonymous KEAP1 mutations. ( d ) Median number of mutations per case by tumor type. Median number of mutations in NRF2-active cases was significantly higher than that in NRF2-inactive cases for the tumor types indicated (*indicates p

    Journal: Scientific Reports

    Article Title: A catalogue of somatic NRF2 gain-of-function mutations in cancer

    doi: 10.1038/s41598-018-31281-0

    Figure Lengend Snippet: NRF2 and KEAP1 mutations are overrepresented in tumors associated with carcinogen exposure. ( a ) Number of cases identified with NRF2 mutation, KEAP1 mutation, or both. ( b ) Percentage of cases within tumor types harboring non-synonymous NRF2 mutations. ( c ) Percentage of cases within tumor types harboring non-synonymous KEAP1 mutations. ( d ) Median number of mutations per case by tumor type. Median number of mutations in NRF2-active cases was significantly higher than that in NRF2-inactive cases for the tumor types indicated (*indicates p

    Article Snippet: Primary antibodies used in this study were raised against β-actin (ACTB) (1:10,000 in milk, Sigma A1978, St. Louis, MO), HA (1:1000 in milk, Cell Signaling, Danvers, MA) MYC (1:1000 in milk, Cell Signaling 2276), and KEAP1 (1:1000 in milk, Cell Signaling 4678).

    Techniques: Mutagenesis

    NRF2 and KEAP1 are preferentially mutated at positions including and beyond NRF2 -DLG and NRF2 -ETGE motifs. ( a ) Number of nonsynonymous mutations at each NRF2 amino acid position from M1 to N605. Labeled amino acids were significantly enriched (p

    Journal: Scientific Reports

    Article Title: A catalogue of somatic NRF2 gain-of-function mutations in cancer

    doi: 10.1038/s41598-018-31281-0

    Figure Lengend Snippet: NRF2 and KEAP1 are preferentially mutated at positions including and beyond NRF2 -DLG and NRF2 -ETGE motifs. ( a ) Number of nonsynonymous mutations at each NRF2 amino acid position from M1 to N605. Labeled amino acids were significantly enriched (p

    Article Snippet: Primary antibodies used in this study were raised against β-actin (ACTB) (1:10,000 in milk, Sigma A1978, St. Louis, MO), HA (1:1000 in milk, Cell Signaling, Danvers, MA) MYC (1:1000 in milk, Cell Signaling 2276), and KEAP1 (1:1000 in milk, Cell Signaling 4678).

    Techniques: Labeling

    Effects of XXT on Nrf2 and Keap1 mRNA expression levels in HUVECs.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

    doi: 10.1155/2015/187265

    Figure Lengend Snippet: Effects of XXT on Nrf2 and Keap1 mRNA expression levels in HUVECs.

    Article Snippet: Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing

    Effects of XXT on the protein expression levels of Keap1, Nrf2, HMOX1, GCLM, and NQO1 in HUVECs.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

    doi: 10.1155/2015/187265

    Figure Lengend Snippet: Effects of XXT on the protein expression levels of Keap1, Nrf2, HMOX1, GCLM, and NQO1 in HUVECs.

    Article Snippet: Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing

    Schematic representation of XXT activities on Keap1-Nrf2-ARE pathway.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

    doi: 10.1155/2015/187265

    Figure Lengend Snippet: Schematic representation of XXT activities on Keap1-Nrf2-ARE pathway.

    Article Snippet: Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques:

    PQ regulates cell proliferation, cell death, and autophagy by modulating Keap1/p65/Nrf2 signal pathway. a , b Western Blot was used to verify the efficiency of p65 overexpression and Nrf2 knock-down in 16HBE cells. c , d The proliferative ability of 16HBE cells treated with 500 μM of PQ for 12 h, 24 h, 48 h, 72 h, and 96 h was detected by CCK-8 assay. e , f The apoptosis ratio of 16HBE cells treated with 150 μM PQ for 2 h was detected by FCM. g – i The autophagy-associated proteins of 16HBE cells treated with 500 μM of PQ for 48 h were detected by Western Blot. Each assay had 3 repetitions (the data are presented as mean ± SD, “*” means statistical significance, p

    Journal: Inflammation

    Article Title: High-Dose Paraquat Induces Human Bronchial 16HBE Cell Death and Aggravates Acute Lung Intoxication in Mice by Regulating Keap1/p65/Nrf2 Signal Pathway

    doi: 10.1007/s10753-018-00956-1

    Figure Lengend Snippet: PQ regulates cell proliferation, cell death, and autophagy by modulating Keap1/p65/Nrf2 signal pathway. a , b Western Blot was used to verify the efficiency of p65 overexpression and Nrf2 knock-down in 16HBE cells. c , d The proliferative ability of 16HBE cells treated with 500 μM of PQ for 12 h, 24 h, 48 h, 72 h, and 96 h was detected by CCK-8 assay. e , f The apoptosis ratio of 16HBE cells treated with 150 μM PQ for 2 h was detected by FCM. g – i The autophagy-associated proteins of 16HBE cells treated with 500 μM of PQ for 48 h were detected by Western Blot. Each assay had 3 repetitions (the data are presented as mean ± SD, “*” means statistical significance, p

    Article Snippet: The primary antibodies of anti-Keap1 (1:1000, #K2769, Sigma, USA), anti-p65 (1:1000, #P0068, Sigma, USA), anti-Nrf2 (1:500, #SAB4501984, Sigma, USA), anti-p21 (1:1000, #SAB4500065, Sigma, USA), anti-Cyclin A2 (1:1000, #C4710, Sigma, USA), anti-Cyclin D1 (1:1000, #C7464, Sigma, USA), anti-Bax (1:500, #B8429, Sigma, USA), anti-Bcl-2 (1:1000, #B3170, Sigma, USA), anti-Caspase 3 (1:500, #C8487, Sigma, USA), anti-LC-3 (1:1000, #8918, Sigma, USA), and anti-β-actin (1:1000, #A5441, Sigma, USA) were employed to be incubated with the membranes overnight at 4 °C.

    Techniques: Western Blot, Over Expression, CCK-8 Assay

    In vivo experiments prove that PQ induced cell intoxication by regulating Keap1/p65/Nrf2 signal pathway. Wild-type C57BL/6 male mice were intraperitoneal injected with saline or 500 μM of PQ and euthanized after 96 h. a , b Western Blot was used to verify and quantify the efficiency of p65 overexpression and Nrf2 knock-out in the lung tissues of the male C57BL/6 mice. c Masson staining was employed to observe the morphologies of the lung tissues of mice treated with PQ (500 μM, 96 h). d The relative mRNA expressions of inflammatory cytokines were detected by real-time qPCR in mice lung tissues and normalized by GAPDH. e The expressions of inflammatory cytokines in the periphery blood of the mice were detected by ELISA. f , g Cell apoptosis-associated proteins were detected and quantified in the mice lung tissues. h , i Cell cycle-associated proteins were detected and quantified in the mice lung tissues. Each assay had at least 3 repetitions (the data are presented as mean ± SD, “*” means statistical significance, p

    Journal: Inflammation

    Article Title: High-Dose Paraquat Induces Human Bronchial 16HBE Cell Death and Aggravates Acute Lung Intoxication in Mice by Regulating Keap1/p65/Nrf2 Signal Pathway

    doi: 10.1007/s10753-018-00956-1

    Figure Lengend Snippet: In vivo experiments prove that PQ induced cell intoxication by regulating Keap1/p65/Nrf2 signal pathway. Wild-type C57BL/6 male mice were intraperitoneal injected with saline or 500 μM of PQ and euthanized after 96 h. a , b Western Blot was used to verify and quantify the efficiency of p65 overexpression and Nrf2 knock-out in the lung tissues of the male C57BL/6 mice. c Masson staining was employed to observe the morphologies of the lung tissues of mice treated with PQ (500 μM, 96 h). d The relative mRNA expressions of inflammatory cytokines were detected by real-time qPCR in mice lung tissues and normalized by GAPDH. e The expressions of inflammatory cytokines in the periphery blood of the mice were detected by ELISA. f , g Cell apoptosis-associated proteins were detected and quantified in the mice lung tissues. h , i Cell cycle-associated proteins were detected and quantified in the mice lung tissues. Each assay had at least 3 repetitions (the data are presented as mean ± SD, “*” means statistical significance, p

    Article Snippet: The primary antibodies of anti-Keap1 (1:1000, #K2769, Sigma, USA), anti-p65 (1:1000, #P0068, Sigma, USA), anti-Nrf2 (1:500, #SAB4501984, Sigma, USA), anti-p21 (1:1000, #SAB4500065, Sigma, USA), anti-Cyclin A2 (1:1000, #C4710, Sigma, USA), anti-Cyclin D1 (1:1000, #C7464, Sigma, USA), anti-Bax (1:500, #B8429, Sigma, USA), anti-Bcl-2 (1:1000, #B3170, Sigma, USA), anti-Caspase 3 (1:500, #C8487, Sigma, USA), anti-LC-3 (1:1000, #8918, Sigma, USA), and anti-β-actin (1:1000, #A5441, Sigma, USA) were employed to be incubated with the membranes overnight at 4 °C.

    Techniques: In Vivo, Mouse Assay, Injection, Western Blot, Over Expression, Knock-Out, Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    PQ’s influences on p65 and the activation of Keap1/Nrf2 signal pathway. a , b , c Real-time qPCR quantification of Keap1, p65, Nrf2 expressions, and normalized by GAPDH, each assay had 10 repetitions. d Western Blot was used to verify the expressions of Keap1, p65, and Nrf2 at protein levels. e Quantification of Keap1, p65, and Nrf2 by ImageJ software according to ( d ). f p65’s impacts on the expressions of Keap1 and Nrf2 were detected by Western Blot and g quantified by ImageJ software. h p65’s effects on Nrf2 downstream targets were detected by Western Blot and i quantified by ImageJ software. j High dose of PQ’s (500 μM) effects on p65, Keap1, and Nrf2 were detected by Western Blot and k quantified by ImageJ software. Each assay had 3 repetitions (the data are presented as mean ±SD, “*” means statistical significance, p

    Journal: Inflammation

    Article Title: High-Dose Paraquat Induces Human Bronchial 16HBE Cell Death and Aggravates Acute Lung Intoxication in Mice by Regulating Keap1/p65/Nrf2 Signal Pathway

    doi: 10.1007/s10753-018-00956-1

    Figure Lengend Snippet: PQ’s influences on p65 and the activation of Keap1/Nrf2 signal pathway. a , b , c Real-time qPCR quantification of Keap1, p65, Nrf2 expressions, and normalized by GAPDH, each assay had 10 repetitions. d Western Blot was used to verify the expressions of Keap1, p65, and Nrf2 at protein levels. e Quantification of Keap1, p65, and Nrf2 by ImageJ software according to ( d ). f p65’s impacts on the expressions of Keap1 and Nrf2 were detected by Western Blot and g quantified by ImageJ software. h p65’s effects on Nrf2 downstream targets were detected by Western Blot and i quantified by ImageJ software. j High dose of PQ’s (500 μM) effects on p65, Keap1, and Nrf2 were detected by Western Blot and k quantified by ImageJ software. Each assay had 3 repetitions (the data are presented as mean ±SD, “*” means statistical significance, p

    Article Snippet: The primary antibodies of anti-Keap1 (1:1000, #K2769, Sigma, USA), anti-p65 (1:1000, #P0068, Sigma, USA), anti-Nrf2 (1:500, #SAB4501984, Sigma, USA), anti-p21 (1:1000, #SAB4500065, Sigma, USA), anti-Cyclin A2 (1:1000, #C4710, Sigma, USA), anti-Cyclin D1 (1:1000, #C7464, Sigma, USA), anti-Bax (1:500, #B8429, Sigma, USA), anti-Bcl-2 (1:1000, #B3170, Sigma, USA), anti-Caspase 3 (1:500, #C8487, Sigma, USA), anti-LC-3 (1:1000, #8918, Sigma, USA), and anti-β-actin (1:1000, #A5441, Sigma, USA) were employed to be incubated with the membranes overnight at 4 °C.

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Software