anti-keap1 Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91
    Thermo Fisher anti keap1
    Effect of DMF and CDDO-Im on intracellular GSH level in <t>Keap1-Mϕ</t> and C151S-Mϕ. Keap1-Mϕ (closed circle) and C151S-Mϕ (open circle) were treated for 24 hours with 1- or 3-µM DMF (A and B), or 30- or 100-nM CDDO-Im,
    Anti Keap1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti keap1/product/Thermo Fisher
    Average 91 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    anti keap1 - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    99
    Millipore keap1 polyclonal antibody
    Effect of DMF and CDDO-Im on intracellular GSH level in <t>Keap1-Mϕ</t> and C151S-Mϕ. Keap1-Mϕ (closed circle) and C151S-Mϕ (open circle) were treated for 24 hours with 1- or 3-µM DMF (A and B), or 30- or 100-nM CDDO-Im,
    Keap1 Polyclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keap1 polyclonal antibody/product/Millipore
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    keap1 polyclonal antibody - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    97
    Abcam anti keap 1 antibody
    Effects of the iodide intake (NI, 10 HI, and 100 HI) on the changes of (a) Nrf2; (b) Keap 1; (c) Srx; and (d) Prx 3 expressions on Days 7, 14, and 28. Representative western blot and histograms of densitometric analyses normalized for the relative  β -actin content. All data is presented as the mean ± SD ( N  = 6 for each group). Statistical analyses were performed by one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD) test.  ∗ p
    Anti Keap 1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti keap 1 antibody/product/Abcam
    Average 97 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    anti keap 1 antibody - by Bioz Stars, 2020-05
    97/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology anti keap1
    Schematic of SILAC-based proteomic mapping of <t>KEAP1</t> modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.
    Anti Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti keap1/product/Santa Cruz Biotechnology
    Average 93 stars, based on 200 article reviews
    Price from $9.99 to $1999.99
    anti keap1 - by Bioz Stars, 2020-05
    93/100 stars
      Buy from Supplier

    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 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti keap1 antibody/product/Abcam
    Average 94 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    anti keap1 antibody - by Bioz Stars, 2020-05
    94/100 stars
      Buy from Supplier

    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 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keap1/product/Cell Signaling Technology Inc
    Average 94 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    keap1 - by Bioz Stars, 2020-05
    94/100 stars
      Buy from Supplier

    90
    Proteintech anti keap1
    Schematic representation of the p62- <t>Keap1-</t> Nrf2 pathway in ovarian cancer cells with the treatment of VK3. Our study provides evidence that p62 promotes Nrf2 signaling through interacting with Keap1, which blocks VK3-induced apoptosis by inhibiting ROS production in SKOV3/DDP cells.
    Anti Keap1, supplied by Proteintech, used in various techniques. Bioz Stars score: 90/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti keap1/product/Proteintech
    Average 90 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    anti keap1 - by Bioz Stars, 2020-05
    90/100 stars
      Buy from Supplier

    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 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti keap1/product/Cell Signaling Technology Inc
    Average 90 stars, based on 98 article reviews
    Price from $9.99 to $1999.99
    anti keap1 - by Bioz Stars, 2020-05
    90/100 stars
      Buy from Supplier

    91
    OriGene 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.
    Keap1, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keap1/product/OriGene
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    keap1 - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    94
    Abcam anti keap1 antibody 1f10b6
    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 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
    https://www.bioz.com/result/anti keap1 antibody 1f10b6/product/Abcam
    Average 94 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    anti keap1 antibody 1f10b6 - by Bioz Stars, 2020-05
    94/100 stars
      Buy from Supplier

    94
    Millipore keap1
    mRNA expression levels (Mean ± SEM) of POU5F1 , CDX2 , NRF2 , <t>KEAP1</t> , BAX , and CASPASE3 in blastocysts treated with Ge-132 during in vitro culture (IVC). Within the same target mRNA, values with different superscript letters (a and b) are significantly (P
    Keap1, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keap1/product/Millipore
    Average 94 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    keap1 - by Bioz Stars, 2020-05
    94/100 stars
      Buy from Supplier

    Image Search Results


    Effect of DMF and CDDO-Im on intracellular GSH level in Keap1-Mϕ and C151S-Mϕ. Keap1-Mϕ (closed circle) and C151S-Mϕ (open circle) were treated for 24 hours with 1- or 3-µM DMF (A and B), or 30- or 100-nM CDDO-Im,

    Journal: Free radical biology & medicine

    Article Title: Validation of the Multiple Sensor Mechanism of the Keap1-Nrf2 System

    doi: 10.1016/j.freeradbiomed.2012.06.023

    Figure Lengend Snippet: Effect of DMF and CDDO-Im on intracellular GSH level in Keap1-Mϕ and C151S-Mϕ. Keap1-Mϕ (closed circle) and C151S-Mϕ (open circle) were treated for 24 hours with 1- or 3-µM DMF (A and B), or 30- or 100-nM CDDO-Im,

    Article Snippet: The samples were subjected to immunoblot analysis using anti-Nrf2 [ ], anti-Keap1 [ ], anti-Lamin B (a nuclear fraction marker; Invitrogen) and anti-Tubulin (a cytoplasmic fraction marker; Sigma) antibodies.

    Techniques:

    Effect of DEM or SFN treatment on Keap1-MEFs and C151S-MEFs (A and E ) Nuclear Nrf2 proteins of Keap1-MEF or C151S-MEF following incubation with 0, 30 and 100 µM DEM (A) or 0, 3 and 10 µM SFN (E) for three hours were examined by Western

    Journal: Free radical biology & medicine

    Article Title: Validation of the Multiple Sensor Mechanism of the Keap1-Nrf2 System

    doi: 10.1016/j.freeradbiomed.2012.06.023

    Figure Lengend Snippet: Effect of DEM or SFN treatment on Keap1-MEFs and C151S-MEFs (A and E ) Nuclear Nrf2 proteins of Keap1-MEF or C151S-MEF following incubation with 0, 30 and 100 µM DEM (A) or 0, 3 and 10 µM SFN (E) for three hours were examined by Western

    Article Snippet: The samples were subjected to immunoblot analysis using anti-Nrf2 [ ], anti-Keap1 [ ], anti-Lamin B (a nuclear fraction marker; Invitrogen) and anti-Tubulin (a cytoplasmic fraction marker; Sigma) antibodies.

    Techniques: Incubation, Western Blot

    Effect of 15d-PGJ2 or ebselen treatment on Keap1-MEFs and C151S-MEFs. (A and E) Nuclear Nrf2 proteins of Keap1-MEFs or C151S-MEFs following incubation with 0, 3 or 10 µµM 15d-PGJ2 (A) or 0, 3 or 10 µM ebselen (E) for three hours

    Journal: Free radical biology & medicine

    Article Title: Validation of the Multiple Sensor Mechanism of the Keap1-Nrf2 System

    doi: 10.1016/j.freeradbiomed.2012.06.023

    Figure Lengend Snippet: Effect of 15d-PGJ2 or ebselen treatment on Keap1-MEFs and C151S-MEFs. (A and E) Nuclear Nrf2 proteins of Keap1-MEFs or C151S-MEFs following incubation with 0, 3 or 10 µµM 15d-PGJ2 (A) or 0, 3 or 10 µM ebselen (E) for three hours

    Article Snippet: The samples were subjected to immunoblot analysis using anti-Nrf2 [ ], anti-Keap1 [ ], anti-Lamin B (a nuclear fraction marker; Invitrogen) and anti-Tubulin (a cytoplasmic fraction marker; Sigma) antibodies.

    Techniques: Incubation

    Effect of DMF, CDDO-Im, CdCl2 or OA-NO2 treatment on Keap1-MEFs or C151S-MEFs. (A, C, E and G) Nuclear Nrf2 proteins in Keap1-MEFs or C151S-MEFs following incubation for 3 hours with 0-, 10- or 30-µM DMF (A), 0-, 30- or 100-nM CDDO-Im (C), 0-,

    Journal: Free radical biology & medicine

    Article Title: Validation of the Multiple Sensor Mechanism of the Keap1-Nrf2 System

    doi: 10.1016/j.freeradbiomed.2012.06.023

    Figure Lengend Snippet: Effect of DMF, CDDO-Im, CdCl2 or OA-NO2 treatment on Keap1-MEFs or C151S-MEFs. (A, C, E and G) Nuclear Nrf2 proteins in Keap1-MEFs or C151S-MEFs following incubation for 3 hours with 0-, 10- or 30-µM DMF (A), 0-, 30- or 100-nM CDDO-Im (C), 0-,

    Article Snippet: The samples were subjected to immunoblot analysis using anti-Nrf2 [ ], anti-Keap1 [ ], anti-Lamin B (a nuclear fraction marker; Invitrogen) and anti-Tubulin (a cytoplasmic fraction marker; Sigma) antibodies.

    Techniques: Incubation

    Effect of DEM, SFN, 15d-PGJ2 or ebselen treatment on peritoneal macrophages derived from Keap1 −/− ::Tg-Keap1 WT or Keap1 −/− ::Tg-Keap1 C151S mice. (A) The experimental scheme for isolation of thioglycollate-elicited peritoneal

    Journal: Free radical biology & medicine

    Article Title: Validation of the Multiple Sensor Mechanism of the Keap1-Nrf2 System

    doi: 10.1016/j.freeradbiomed.2012.06.023

    Figure Lengend Snippet: Effect of DEM, SFN, 15d-PGJ2 or ebselen treatment on peritoneal macrophages derived from Keap1 −/− ::Tg-Keap1 WT or Keap1 −/− ::Tg-Keap1 C151S mice. (A) The experimental scheme for isolation of thioglycollate-elicited peritoneal

    Article Snippet: The samples were subjected to immunoblot analysis using anti-Nrf2 [ ], anti-Keap1 [ ], anti-Lamin B (a nuclear fraction marker; Invitrogen) and anti-Tubulin (a cytoplasmic fraction marker; Sigma) antibodies.

    Techniques: Derivative Assay, Mouse Assay, Isolation

    Knockdown of KPNA6 inhibits nuclear import of Keap1. (A) NIH 3T3 cells were cotransfected with either scrambled control siRNA or KPNA6 siRNA, along with an expression vector for Keap1. At 48 h after transfection, cells were treated with 5 nM LMB for the

    Journal: Molecular and Cellular Biology

    Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿

    doi: 10.1128/MCB.05036-11

    Figure Lengend Snippet: Knockdown of KPNA6 inhibits nuclear import of Keap1. (A) NIH 3T3 cells were cotransfected with either scrambled control siRNA or KPNA6 siRNA, along with an expression vector for Keap1. At 48 h after transfection, cells were treated with 5 nM LMB for the

    Article Snippet: The rabbit anti-Keap1 antibody was generated in rabbits challenged with purified full-length His-tagged human Keap1 protein, followed by purification on a column with immobilized His-Keap1 protein (Pierce).

    Techniques: Expressing, Plasmid Preparation, Transfection

    Overexpression of KPNA6 attenuates the inducible Nrf2 signaling in response to oxidative stress. (A) KPNA6 decreases the inducible Nrf2 protein level and the expression of its downstream detoxification genes without altering Keap1 protein levels. HEK293T

    Journal: Molecular and Cellular Biology

    Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿

    doi: 10.1128/MCB.05036-11

    Figure Lengend Snippet: Overexpression of KPNA6 attenuates the inducible Nrf2 signaling in response to oxidative stress. (A) KPNA6 decreases the inducible Nrf2 protein level and the expression of its downstream detoxification genes without altering Keap1 protein levels. HEK293T

    Article Snippet: The rabbit anti-Keap1 antibody was generated in rabbits challenged with purified full-length His-tagged human Keap1 protein, followed by purification on a column with immobilized His-Keap1 protein (Pierce).

    Techniques: Over Expression, Expressing

    The C-terminal Kelch domain of Keap1 mediates its nuclear entry. (A) Schematic of conserved domains in human Keap1 protein. Keap1 contains an N-terminal BTB domain, a C-terminal Kelch domain, and a linker region in between the two domains. The nuclear

    Journal: Molecular and Cellular Biology

    Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿

    doi: 10.1128/MCB.05036-11

    Figure Lengend Snippet: The C-terminal Kelch domain of Keap1 mediates its nuclear entry. (A) Schematic of conserved domains in human Keap1 protein. Keap1 contains an N-terminal BTB domain, a C-terminal Kelch domain, and a linker region in between the two domains. The nuclear

    Article Snippet: The rabbit anti-Keap1 antibody was generated in rabbits challenged with purified full-length His-tagged human Keap1 protein, followed by purification on a column with immobilized His-Keap1 protein (Pierce).

    Techniques:

    Schematic model of Nrf2 regulation by Keap1. Keap1 is a key regulator of the Nrf2-signaling pathway and serves as a molecular switch to turn the Nrf2-mediated antioxidant response on and off. (1) Oxidative stress or chemopreventive compounds cause a conformational

    Journal: Molecular and Cellular Biology

    Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿

    doi: 10.1128/MCB.05036-11

    Figure Lengend Snippet: Schematic model of Nrf2 regulation by Keap1. Keap1 is a key regulator of the Nrf2-signaling pathway and serves as a molecular switch to turn the Nrf2-mediated antioxidant response on and off. (1) Oxidative stress or chemopreventive compounds cause a conformational

    Article Snippet: The rabbit anti-Keap1 antibody was generated in rabbits challenged with purified full-length His-tagged human Keap1 protein, followed by purification on a column with immobilized His-Keap1 protein (Pierce).

    Techniques:

    The nuclear import of Keap1 occurs at its physiological protein level and is not dependent on Nrf1 or Nrf2. (A) GFP-tagged Keap1 proteins were expressed at levels similar to the levels of endogenous Keap1 in the stable MEF cell lines. Keap1 −/−

    Journal: Molecular and Cellular Biology

    Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿

    doi: 10.1128/MCB.05036-11

    Figure Lengend Snippet: The nuclear import of Keap1 occurs at its physiological protein level and is not dependent on Nrf1 or Nrf2. (A) GFP-tagged Keap1 proteins were expressed at levels similar to the levels of endogenous Keap1 in the stable MEF cell lines. Keap1 −/−

    Article Snippet: The rabbit anti-Keap1 antibody was generated in rabbits challenged with purified full-length His-tagged human Keap1 protein, followed by purification on a column with immobilized His-Keap1 protein (Pierce).

    Techniques:

    KPNA6 interacts with the Kelch domain of Keap1. (A) KPNA6 binds Keap1 in vitro . Purified His-tagged Keap1 proteins were incubated with the indicated in vitro -translated (IVT) 35 S-labeled proteins of the importin family, followed by pulldown with Ni-NTA

    Journal: Molecular and Cellular Biology

    Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿

    doi: 10.1128/MCB.05036-11

    Figure Lengend Snippet: KPNA6 interacts with the Kelch domain of Keap1. (A) KPNA6 binds Keap1 in vitro . Purified His-tagged Keap1 proteins were incubated with the indicated in vitro -translated (IVT) 35 S-labeled proteins of the importin family, followed by pulldown with Ni-NTA

    Article Snippet: The rabbit anti-Keap1 antibody was generated in rabbits challenged with purified full-length His-tagged human Keap1 protein, followed by purification on a column with immobilized His-Keap1 protein (Pierce).

    Techniques: In Vitro, Purification, Incubation, Labeling

    Overexpression of KPNA6 facilitates nuclear import of Keap1. (A) NIH 3T3 cells were transfected with an expression vector for Keap1 with or without a vector for Myc-tagged KPNA6. Cells were treated with 5 nM LMB for the indicated times. After fixation

    Journal: Molecular and Cellular Biology

    Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿

    doi: 10.1128/MCB.05036-11

    Figure Lengend Snippet: Overexpression of KPNA6 facilitates nuclear import of Keap1. (A) NIH 3T3 cells were transfected with an expression vector for Keap1 with or without a vector for Myc-tagged KPNA6. Cells were treated with 5 nM LMB for the indicated times. After fixation

    Article Snippet: The rabbit anti-Keap1 antibody was generated in rabbits challenged with purified full-length His-tagged human Keap1 protein, followed by purification on a column with immobilized His-Keap1 protein (Pierce).

    Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation

    Keap1-dependent ubiquitination of Nrf2 requires C273. (A) MDA-MB-231 cells were cotransfected with an expression vector for HA-ubiquitin (HA-Ub) (lanes 1 to 10), a Gal4-Neh2 fusion protein (lanes 3 to 10), or expression vectors for either wild-type (WT) Keap1 (lanes 5 and 6), Keap1-C273S (lanes 7 and 8), or Keap1-C151S (lanes 9 and 10). The transfected cells were exposed to DMSO (odd-numbered lanes) or MG132 (even-numbered lanes) for 5 h. Cells were lysed in 2% SDS and 10 mM NEM to block ubiquitin hydrolase activity. Anti-Gal4 (α-Gal) immunoprecipitates (IP) were analyzed by immunoblot with anti-HA (α-HA) antibodies. IgG, immunoglobulin G. (B) MDA-MB-231 cells were transfected with expression plasmids and treated with MG132 as described for panel A, except that no HA-ubiquitin plasmids were transfected. Total cell lysates were collected in sample buffer and subjected to immunoblot analysis with anti-Gal antibodies. (C) MDA-MB-231 cells were cotransfected with expression vectors for HA-ubiquitin and Nrf2 (lanes 1 to 6), and expression vectors for either wild-type Keap1 (lanes 1 and 2), Keap1-C273S (lanes 3 and 4), or Keap1-C151S (lanes 5 and 6). The transfected cells were exposed to DMSO (odd-numbered lanes) or MG132 (even-numbered lanes) for 5 h. Cells were lysed in 2% SDS and 10 mM NEM to block ubiquitin hydrolase activity. Anti-Nrf2 (α-Nrf2) immunoprecipitates were analyzed by immunoblot with anti-HA antibodies. (D) MDA-MB-231 cells were cotransfected with an expression vector for HA-Nrf2 (lanes 1 to 6) and for either wild-type Keap1 (lanes 1 and 2), Keap1-C273S (lanes 3 and 4), or Keap1-C151S (lanes 5 and 6). The transfected cells were exposed to DMSO (odd-numbered lanes) or MG132 (even-numbered lanes) for 5 h. Total cell lysates were collected in sample buffer and subjected to immunoblot analysis with anti-HA antibodies.

    Journal: Molecular and Cellular Biology

    Article Title: Distinct Cysteine Residues in Keap1 Are Required for Keap1-Dependent Ubiquitination of Nrf2 and for Stabilization of Nrf2 by Chemopreventive Agents and Oxidative Stress

    doi: 10.1128/MCB.23.22.8137-8151.2003

    Figure Lengend Snippet: Keap1-dependent ubiquitination of Nrf2 requires C273. (A) MDA-MB-231 cells were cotransfected with an expression vector for HA-ubiquitin (HA-Ub) (lanes 1 to 10), a Gal4-Neh2 fusion protein (lanes 3 to 10), or expression vectors for either wild-type (WT) Keap1 (lanes 5 and 6), Keap1-C273S (lanes 7 and 8), or Keap1-C151S (lanes 9 and 10). The transfected cells were exposed to DMSO (odd-numbered lanes) or MG132 (even-numbered lanes) for 5 h. Cells were lysed in 2% SDS and 10 mM NEM to block ubiquitin hydrolase activity. Anti-Gal4 (α-Gal) immunoprecipitates (IP) were analyzed by immunoblot with anti-HA (α-HA) antibodies. IgG, immunoglobulin G. (B) MDA-MB-231 cells were transfected with expression plasmids and treated with MG132 as described for panel A, except that no HA-ubiquitin plasmids were transfected. Total cell lysates were collected in sample buffer and subjected to immunoblot analysis with anti-Gal antibodies. (C) MDA-MB-231 cells were cotransfected with expression vectors for HA-ubiquitin and Nrf2 (lanes 1 to 6), and expression vectors for either wild-type Keap1 (lanes 1 and 2), Keap1-C273S (lanes 3 and 4), or Keap1-C151S (lanes 5 and 6). The transfected cells were exposed to DMSO (odd-numbered lanes) or MG132 (even-numbered lanes) for 5 h. Cells were lysed in 2% SDS and 10 mM NEM to block ubiquitin hydrolase activity. Anti-Nrf2 (α-Nrf2) immunoprecipitates were analyzed by immunoblot with anti-HA antibodies. (D) MDA-MB-231 cells were cotransfected with an expression vector for HA-Nrf2 (lanes 1 to 6) and for either wild-type Keap1 (lanes 1 and 2), Keap1-C273S (lanes 3 and 4), or Keap1-C151S (lanes 5 and 6). The transfected cells were exposed to DMSO (odd-numbered lanes) or MG132 (even-numbered lanes) for 5 h. Total cell lysates were collected in sample buffer and subjected to immunoblot analysis with anti-HA antibodies.

    Article Snippet: Antibodies were generated in rabbits against the full-length Keap1 protein and anti-Keap1 immunoglobulin G was purified over a column containing immobilized His-Keap1 protein with reagents obtained from Pierce.

    Techniques: Multiple Displacement Amplification, Expressing, Plasmid Preparation, Transfection, Blocking Assay, Activity Assay

    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

    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:

    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

    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

    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

    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:

    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

    Schematic of SILAC-based proteomic mapping of KEAP1 modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: Schematic of SILAC-based proteomic mapping of KEAP1 modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

    Techniques: Nuclear Magnetic Resonance, Labeling, Cell Culture, Expressing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Modification, Western Blot, Isolation, Incubation

    Modulation of PGK1 induces HMW-KEAP1. a, Anti-pgK (phosphoglyceryl-lysine) and anti-GAPDH Western blots analysis of CBR-470-1 or DMSO-treated IMR32 cells at early (30 min) and late (24 hr) time points ( n =6). b, Anti-FLAG (left) and anti-pgK (right) Western blot analysis of affinity purified FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 30 min. Duplicate samples were run under non-reducing (left) and reducing (DTT, right) conditions (n=6). c, Densitometry quantification of total endogenous KEAP1 levels (combined bands at ∼70 and 140 kDa) in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). d , Western blot detection of FLAG-KEAP1 in HEK293T cells comparing no-reducing reagent to DTT (left), and stability of CBR-470-1-dependent HMW-KEAP1 to the presence of DTT (12.5 mM final concentration, middle) and beta-mercaptoethanol (5% v/v final concentration, right) during sample preparation. treated with DMSO or CBR-470-1 for 8 hours ( n =8). e, Time-dependent CBR-470-1 treatment of HEK293T cells expressing FLAG-KEAP1. Time-dependent assays were run with 20 μM CBR-470-1 with Western blot analysis at the indicated time-points ( n =8). f, g, Western blot detection ( f ) and quantification ( g ) of endogenous KEAP1 and β-actin in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). Arrows indicate monomeric (∼70 kDa) and HMW-KEAP1 (∼140 kDa) bands. h, i, Western blot ( h ) detection and quantification ( i ) of FLAG-KEAP1 in HEK293T cells exposed to increasing doses of CBR-470-1 ( n =3). j, Kinetic qRT-PCR measurement of NQO1 mRNA levels from IMR32 cells treated with tBHQ (10 μM) or CBR-470-1 (10 μM) for the indicated times ( n =3). k, Quantification of HMW-KEAP1 formation upon treatment with CBR-470-1 or the direct KEAP1 alkylator TBHQ, in the presence or absence of reduced glutathione (GSH) or N -acetylcysteine (NAC) ( n =3). All measurements taken after 8 hour of treatment in FLAG-KEAP1 expressing HEK293T cells. l, Transient shRNA knockdown of PGK1 induced HMW-KEAP1 formation, which was blocked by co-treatment of cells by GSH ( n =3). m, Anti-FLAG Western blot analysis of FLAG-KEAP1 monomer and HMW-KEAP1 fraction with dose-dependent incubation of distilled MGx in lysate from HEK-293T cells expressing FLAG-KEAP1 ( n =4). n, SDS-PAGE gel (silver stain) and anti-FLAG Western blot analysis of purified KEAP1 treated with the MGx under the indicated reducing conditions for 2 hr at 37°C ( n =3). Purified protein reactions were quenched in 4x SDS loading buffer containing βME and processed for gel analysis as in (d). Data shown represent mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: Modulation of PGK1 induces HMW-KEAP1. a, Anti-pgK (phosphoglyceryl-lysine) and anti-GAPDH Western blots analysis of CBR-470-1 or DMSO-treated IMR32 cells at early (30 min) and late (24 hr) time points ( n =6). b, Anti-FLAG (left) and anti-pgK (right) Western blot analysis of affinity purified FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 30 min. Duplicate samples were run under non-reducing (left) and reducing (DTT, right) conditions (n=6). c, Densitometry quantification of total endogenous KEAP1 levels (combined bands at ∼70 and 140 kDa) in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). d , Western blot detection of FLAG-KEAP1 in HEK293T cells comparing no-reducing reagent to DTT (left), and stability of CBR-470-1-dependent HMW-KEAP1 to the presence of DTT (12.5 mM final concentration, middle) and beta-mercaptoethanol (5% v/v final concentration, right) during sample preparation. treated with DMSO or CBR-470-1 for 8 hours ( n =8). e, Time-dependent CBR-470-1 treatment of HEK293T cells expressing FLAG-KEAP1. Time-dependent assays were run with 20 μM CBR-470-1 with Western blot analysis at the indicated time-points ( n =8). f, g, Western blot detection ( f ) and quantification ( g ) of endogenous KEAP1 and β-actin in IMR32 cells treated with DMSO or CBR-470-1 for the indicated times ( n =6). Arrows indicate monomeric (∼70 kDa) and HMW-KEAP1 (∼140 kDa) bands. h, i, Western blot ( h ) detection and quantification ( i ) of FLAG-KEAP1 in HEK293T cells exposed to increasing doses of CBR-470-1 ( n =3). j, Kinetic qRT-PCR measurement of NQO1 mRNA levels from IMR32 cells treated with tBHQ (10 μM) or CBR-470-1 (10 μM) for the indicated times ( n =3). k, Quantification of HMW-KEAP1 formation upon treatment with CBR-470-1 or the direct KEAP1 alkylator TBHQ, in the presence or absence of reduced glutathione (GSH) or N -acetylcysteine (NAC) ( n =3). All measurements taken after 8 hour of treatment in FLAG-KEAP1 expressing HEK293T cells. l, Transient shRNA knockdown of PGK1 induced HMW-KEAP1 formation, which was blocked by co-treatment of cells by GSH ( n =3). m, Anti-FLAG Western blot analysis of FLAG-KEAP1 monomer and HMW-KEAP1 fraction with dose-dependent incubation of distilled MGx in lysate from HEK-293T cells expressing FLAG-KEAP1 ( n =4). n, SDS-PAGE gel (silver stain) and anti-FLAG Western blot analysis of purified KEAP1 treated with the MGx under the indicated reducing conditions for 2 hr at 37°C ( n =3). Purified protein reactions were quenched in 4x SDS loading buffer containing βME and processed for gel analysis as in (d). Data shown represent mean ± SEM of biologically independent samples.

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

    Techniques: Western Blot, Affinity Purification, Concentration Assay, Sample Prep, Expressing, Quantitative RT-PCR, shRNA, Incubation, SDS Page, Silver Staining, Purification

    Methylglyoxal modifies KEAP1 to form a covalent, high molecular weight dimer and activate NRF2 signaling. a, Time-course, anti-FLAG Western blot analysis of whole cell lysates from HEK293T cells expressing FLAG-KEAP1 treated with DMSO or CBR-470-1. b, Western blot monitoring of FLAG-KEAP1 migration in HEK293T lysates after incubation with central glycolytic metabolites in vitro (1 and 5 mM, left and right for each metabolite). c, FLAG-KEAP1 (red) and β-actin (green) from HEK293T cells treated with MGx (5 mM) for 8 hr. d, Relative NQO1 and HMOX1 mRNA levels in IMR32 cells treated with MGx (1 mM) or water control ( n =3). e, LC-MS/MS quantitation of cellular MGx levels in IMR32 cells treated with CBR-470-1 relative to DMSO ( n =4). f, ARE-LUC reporter activity in HEK293T cells with transient shRNA knockdown of GLO1 ( n =8). Univariate two-sided t-test ( d, f ); data are mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: Methylglyoxal modifies KEAP1 to form a covalent, high molecular weight dimer and activate NRF2 signaling. a, Time-course, anti-FLAG Western blot analysis of whole cell lysates from HEK293T cells expressing FLAG-KEAP1 treated with DMSO or CBR-470-1. b, Western blot monitoring of FLAG-KEAP1 migration in HEK293T lysates after incubation with central glycolytic metabolites in vitro (1 and 5 mM, left and right for each metabolite). c, FLAG-KEAP1 (red) and β-actin (green) from HEK293T cells treated with MGx (5 mM) for 8 hr. d, Relative NQO1 and HMOX1 mRNA levels in IMR32 cells treated with MGx (1 mM) or water control ( n =3). e, LC-MS/MS quantitation of cellular MGx levels in IMR32 cells treated with CBR-470-1 relative to DMSO ( n =4). f, ARE-LUC reporter activity in HEK293T cells with transient shRNA knockdown of GLO1 ( n =8). Univariate two-sided t-test ( d, f ); data are mean ± SEM of biologically independent samples.

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

    Techniques: Molecular Weight, Western Blot, Expressing, Migration, Incubation, In Vitro, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Quantitation Assay, Activity Assay, shRNA

    Methylglyoxal forms a novel posttranslational modification between proximal cysteine and arginine residues in KEAP1. a, Quantified HMW-KEAP1 formation of wild-type or mutant FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 8 hr ( n =23 for WT; n =16 for R15A; n =13 for C151S; n =7 for K39R, R135A; n =4 for R6A, R50A, all other C-to-S mutations, and R15/135A C151S triple-mutant; n =3 for R15/135A, and all K-to-M mutations). b, Schematic of the model peptide screen for intramolecular modifications formed by MGx and nucleophilic residues. c, Total ion- (TIC) and extracted ion chromatograms (EIC) from MGx- and mock-treated peptide, with a new peak in the former condition marked with an asterisk. EICs are specific to the indicated m/ z . ( n =3 independent biological replicates). d, 1 H-NMR spectra of the unmodified (top) and MICA-modified (bottom) model peptide, with pertinent protons highlighted in each. Notable changes in the MICA-modified spectrum include the appearance of a singlet at 2.04 p.p.m. (allyl methyl in MICA), loss of the thiol proton at 2.43 p.p.m., and changes in chemical shift and splitting pattern of the cysteine beta protons and the arginine delta and epsilon protons. Full spectra and additional multidimensional NMR spectra can be found in Extended Data Fig. 7 . e, EIC from LC-MS/MS analyses of gel-isolated and digested HMW-KEAP1 (CBR-470-1 and MGx-induced) and monomeric KEAP1 for the C151-R135 crosslinked peptide. Slight retention time variation was observed on commercial columns ( n= 3 independent biological replicates). f, PRM chromatograms for the parent and six parent-to-daughter transitions in representative targeted proteomic runs from HMW-KEAP1 and monomeric digests ( n =6). g, Schematic depicting the direct communication between glucose metabolism and KEAP1-NRF2 signaling mediated by MGx modification of KEAP1 and subsequent activation of the NRF2 transcriptional program. Univariate two-sided t-test ( a ); data are mean ± SEM of biologically independent samples.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: Methylglyoxal forms a novel posttranslational modification between proximal cysteine and arginine residues in KEAP1. a, Quantified HMW-KEAP1 formation of wild-type or mutant FLAG-KEAP1 from HEK293T cells treated with DMSO or CBR-470-1 for 8 hr ( n =23 for WT; n =16 for R15A; n =13 for C151S; n =7 for K39R, R135A; n =4 for R6A, R50A, all other C-to-S mutations, and R15/135A C151S triple-mutant; n =3 for R15/135A, and all K-to-M mutations). b, Schematic of the model peptide screen for intramolecular modifications formed by MGx and nucleophilic residues. c, Total ion- (TIC) and extracted ion chromatograms (EIC) from MGx- and mock-treated peptide, with a new peak in the former condition marked with an asterisk. EICs are specific to the indicated m/ z . ( n =3 independent biological replicates). d, 1 H-NMR spectra of the unmodified (top) and MICA-modified (bottom) model peptide, with pertinent protons highlighted in each. Notable changes in the MICA-modified spectrum include the appearance of a singlet at 2.04 p.p.m. (allyl methyl in MICA), loss of the thiol proton at 2.43 p.p.m., and changes in chemical shift and splitting pattern of the cysteine beta protons and the arginine delta and epsilon protons. Full spectra and additional multidimensional NMR spectra can be found in Extended Data Fig. 7 . e, EIC from LC-MS/MS analyses of gel-isolated and digested HMW-KEAP1 (CBR-470-1 and MGx-induced) and monomeric KEAP1 for the C151-R135 crosslinked peptide. Slight retention time variation was observed on commercial columns ( n= 3 independent biological replicates). f, PRM chromatograms for the parent and six parent-to-daughter transitions in representative targeted proteomic runs from HMW-KEAP1 and monomeric digests ( n =6). g, Schematic depicting the direct communication between glucose metabolism and KEAP1-NRF2 signaling mediated by MGx modification of KEAP1 and subsequent activation of the NRF2 transcriptional program. Univariate two-sided t-test ( a ); data are mean ± SEM of biologically independent samples.

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

    Techniques: Modification, Mutagenesis, Nuclear Magnetic Resonance, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Isolation, Activation Assay

    MS2 analysis of CR-MGx crosslinked KEAP1 peptide. a, Targeted Parallel reaction monitoring (PRM) transitions ( n =6). b, Annotated MS2 spectrum from the crosslinked C151-R135 KEAP1 peptide.

    Journal: Nature

    Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling

    doi: 10.1038/s41586-018-0622-0

    Figure Lengend Snippet: MS2 analysis of CR-MGx crosslinked KEAP1 peptide. a, Targeted Parallel reaction monitoring (PRM) transitions ( n =6). b, Annotated MS2 spectrum from the crosslinked C151-R135 KEAP1 peptide.

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

    Techniques:

    Effects of oleanolic acid (OA) on nuclear/total Nrf2, Keap1, HO-1 and NQO1 expressions in chronic CsA nephropathy. (A) Representative Western blot showing the effects of OA on nuclear/total Nrf2, Keap1, HO-1 and NQO1 expression in chronic CsA nephropathy. (B) Quantitative analyses for total Nrf2/β-actin. There were no significant differences identified by quantitative analysis for immunoblotting of total Nrf2 among the experimental groups. (C) Quantitative analyses for nuclear/total Nrf2. There was increased nuclear/total Nrf2 in the CsA + OA compared with CsA. *p

    Journal: Journal of Translational Medicine

    Article Title: Delayed treatment with oleanolic acid attenuates tubulointerstitial fibrosis in chronic cyclosporine nephropathy through Nrf2/HO-1 signaling

    doi: 10.1186/1479-5876-12-50

    Figure Lengend Snippet: Effects of oleanolic acid (OA) on nuclear/total Nrf2, Keap1, HO-1 and NQO1 expressions in chronic CsA nephropathy. (A) Representative Western blot showing the effects of OA on nuclear/total Nrf2, Keap1, HO-1 and NQO1 expression in chronic CsA nephropathy. (B) Quantitative analyses for total Nrf2/β-actin. There were no significant differences identified by quantitative analysis for immunoblotting of total Nrf2 among the experimental groups. (C) Quantitative analyses for nuclear/total Nrf2. There was increased nuclear/total Nrf2 in the CsA + OA compared with CsA. *p

    Article Snippet: Specifically, proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and detected with the following antibody concentrations: Nrf2 (1:1000; Santa Cruz Biotechnology Inc, Texas, USA), Keap1 (1:1000; Santa Cruz Biotechnology Inc, Texas, USA), HO-1 (1:1000; BD Biosciences, California, USA), NQO1 (1:1000; Santa Cruz Biotechnology Inc, Texas, USA), Bcl-2 (1:500; Santa Cruz Biotechnology Inc, Texas, USA), Bax (1:500; Santa Cruz Biotechnology Inc, Texas, USA), SOD1 (1:5000; Assay Designs, MI, USA), SOD2 (1:10000; Abcam, Cambridge, UK), Catalase (1:2000; Abcam, Cambridge, UK), and β-actin (1:10000; Sigma-Aldrich, MO, USA).

    Techniques: Western Blot, Expressing

    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

    Schematic representation of the p62- Keap1- Nrf2 pathway in ovarian cancer cells with the treatment of VK3. Our study provides evidence that p62 promotes Nrf2 signaling through interacting with Keap1, which blocks VK3-induced apoptosis by inhibiting ROS production in SKOV3/DDP cells.

    Journal: Journal of Cancer

    Article Title: p62 Suppressed VK3-induced Oxidative Damage Through Keap1/Nrf2 Pathway In Human Ovarian Cancer Cells

    doi: 10.7150/jca.34423

    Figure Lengend Snippet: Schematic representation of the p62- Keap1- Nrf2 pathway in ovarian cancer cells with the treatment of VK3. Our study provides evidence that p62 promotes Nrf2 signaling through interacting with Keap1, which blocks VK3-induced apoptosis by inhibiting ROS production in SKOV3/DDP cells.

    Article Snippet: Cells were then incubated with primary antibodies anti-p62 and anti-Keap1 (both at 1:100 dilution in PBS) overnight at 4°C.

    Techniques:

    The interaction between p62 and Keap1 increased with VK3 treatment in SKOV3/DDP cells. (A) Both cells were treated with 15 µM VK3 for 8 or 16 h. Cell lysates were subjected to immunoblot analysis with anti-p62. (B) The expression of p62 in (A) was quantified. Data are presented as mean ± SD, n = 3. * P

    Journal: Journal of Cancer

    Article Title: p62 Suppressed VK3-induced Oxidative Damage Through Keap1/Nrf2 Pathway In Human Ovarian Cancer Cells

    doi: 10.7150/jca.34423

    Figure Lengend Snippet: The interaction between p62 and Keap1 increased with VK3 treatment in SKOV3/DDP cells. (A) Both cells were treated with 15 µM VK3 for 8 or 16 h. Cell lysates were subjected to immunoblot analysis with anti-p62. (B) The expression of p62 in (A) was quantified. Data are presented as mean ± SD, n = 3. * P

    Article Snippet: Cells were then incubated with primary antibodies anti-p62 and anti-Keap1 (both at 1:100 dilution in PBS) overnight at 4°C.

    Techniques: Expressing

    In vivo analyses of Sqstm1-GFP KI/+ mice under autophagy-impaired conditions. (A) Immunoblotting of livers of mice of the indicated genotypes. Liver homogenates prepared from 4–5-week-old mice of the indicated genotypes were subjected to NuPAGE and analyzed by immunoblotting with the indicated antibodies. (B) Immunohistofluorescence analysis of Sqstm1–GFP, Sqstm1, Ser351-phosphorylated Sqstm1 and Keap1. Liver sections from 4–5-week-old mice of the indicated genotypes were triple-immunostained for GFP, Sqstm1, and Ser351-phosphorylated Sqstm1 (upper panels) or GFP, Sqstm1 and Keap1 antibodies (bottom panels). The rightmost column shows the merged images of GFP (green), Sqstm1 (red), and S351-phosphorylated Sqstm1 (blue) in the upper panel, and GFP (green), Sqstm1 (red), and Keap1 (blue) in the bottom panel. Scale bars: 50 µm. (C) Quantitative real-time PCR analyses of Nqo1 , Gstm1 , and Ho-1 in mouse livers. Total RNAs were prepared from livers of 4–5-week-old mice of the indicated genotypes. Values were normalized to the amount of mRNA in the Atg7 f/f liver. Experiments were performed three times. Data are means±s.e.m. * P

    Journal: Journal of Cell Science

    Article Title: Sqstm1–GFP knock-in mice reveal dynamic actions of Sqstm1 during autophagy and under stress conditions in living cells

    doi: 10.1242/jcs.180174

    Figure Lengend Snippet: In vivo analyses of Sqstm1-GFP KI/+ mice under autophagy-impaired conditions. (A) Immunoblotting of livers of mice of the indicated genotypes. Liver homogenates prepared from 4–5-week-old mice of the indicated genotypes were subjected to NuPAGE and analyzed by immunoblotting with the indicated antibodies. (B) Immunohistofluorescence analysis of Sqstm1–GFP, Sqstm1, Ser351-phosphorylated Sqstm1 and Keap1. Liver sections from 4–5-week-old mice of the indicated genotypes were triple-immunostained for GFP, Sqstm1, and Ser351-phosphorylated Sqstm1 (upper panels) or GFP, Sqstm1 and Keap1 antibodies (bottom panels). The rightmost column shows the merged images of GFP (green), Sqstm1 (red), and S351-phosphorylated Sqstm1 (blue) in the upper panel, and GFP (green), Sqstm1 (red), and Keap1 (blue) in the bottom panel. Scale bars: 50 µm. (C) Quantitative real-time PCR analyses of Nqo1 , Gstm1 , and Ho-1 in mouse livers. Total RNAs were prepared from livers of 4–5-week-old mice of the indicated genotypes. Values were normalized to the amount of mRNA in the Atg7 f/f liver. Experiments were performed three times. Data are means±s.e.m. * P

    Article Snippet: For immunolabeling, sections were processed for antigen retrieval in Immunosaver (Nissin EM, Tokyo, Japan) using a microwave processor (Azumaya Corporation, Tokyo, Japan), followed by blocking and incubation for 2–3 days at 4°C with three primary antibodies; mouse monoclonal antibody against GFP (clone 7.1/13.1, Roche), guinea pig polyclonal antibody against Sqstm1 (GP62-C, Progen Biotechnik, Heidelberg, Germany), and rabbit polyclonal antibody against Ser351-phosphorylated Sqstm1 ( ) or Keap1 (Proteintech Group).

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

    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

    mRNA expression levels (Mean ± SEM) of POU5F1 , CDX2 , NRF2 , KEAP1 , BAX , and CASPASE3 in blastocysts treated with Ge-132 during in vitro culture (IVC). Within the same target mRNA, values with different superscript letters (a and b) are significantly (P

    Journal: The Journal of Reproduction and Development

    Article Title: Carboxyethylgermanium sesquioxide (Ge-132) treatment during in vitro culture protects fertilized porcine embryos against oxidative stress induced apoptosis

    doi: 10.1262/jrd.2017-020

    Figure Lengend Snippet: mRNA expression levels (Mean ± SEM) of POU5F1 , CDX2 , NRF2 , KEAP1 , BAX , and CASPASE3 in blastocysts treated with Ge-132 during in vitro culture (IVC). Within the same target mRNA, values with different superscript letters (a and b) are significantly (P

    Article Snippet: The primary antibodies used were KEAP1 (ABS97, Millipore, Temecula, CA, 1:200) and BCL2 (SC23960, Santa Cruz, Dallas, TX, 1:200).

    Techniques: Expressing, In Vitro

    Effect of Ge-132 on the protein expression of KEAP1 and BCL2 in blastocysts treated with Ge-132 during in vitro culture (IVC). (A) Green fluorescence signals reveal KEAP1 protein localization, while red fluorescence signals showed BCL2 protein expression. The cells were counterstained with Hoechst (blue fluorescence) to visualize nuclear morphology. The white arrow indicates the absent of KEAP1 in nuclei (localization in cytosol). Each image shown is representative of at least 14 random fields observed. Indicated scale bars signify 100 µm distance. (B) The intensity of KEAP1 and BCL2 was quantitated by densitometric analysis and is shown in the histogram. The data is represented as the mean ± SD from three independent experiments. Within each group end point (KEAP1 and BCL2), the bars with different letters (a, b) are significantly (P

    Journal: The Journal of Reproduction and Development

    Article Title: Carboxyethylgermanium sesquioxide (Ge-132) treatment during in vitro culture protects fertilized porcine embryos against oxidative stress induced apoptosis

    doi: 10.1262/jrd.2017-020

    Figure Lengend Snippet: Effect of Ge-132 on the protein expression of KEAP1 and BCL2 in blastocysts treated with Ge-132 during in vitro culture (IVC). (A) Green fluorescence signals reveal KEAP1 protein localization, while red fluorescence signals showed BCL2 protein expression. The cells were counterstained with Hoechst (blue fluorescence) to visualize nuclear morphology. The white arrow indicates the absent of KEAP1 in nuclei (localization in cytosol). Each image shown is representative of at least 14 random fields observed. Indicated scale bars signify 100 µm distance. (B) The intensity of KEAP1 and BCL2 was quantitated by densitometric analysis and is shown in the histogram. The data is represented as the mean ± SD from three independent experiments. Within each group end point (KEAP1 and BCL2), the bars with different letters (a, b) are significantly (P

    Article Snippet: The primary antibodies used were KEAP1 (ABS97, Millipore, Temecula, CA, 1:200) and BCL2 (SC23960, Santa Cruz, Dallas, TX, 1:200).

    Techniques: Expressing, In Vitro, Fluorescence