anti-keap1 antibody Search Results


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  • 92
    Millipore anti keap1 ab1 antibody
    Anti Keap1 Ab1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam keap1 antibody
    Chrysin deactivates Nrf2 signaling pathway in a <t>Keap1-independent</t> 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
    Keap1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gapdh  (Abcam)
    87
    Abcam gapdh
    <t>NRF2</t> regulates KEAP1, NQO1, GCLC and HO1 in MRC-5 cells treated with laminarin. MRC-5 cells were transfected with the overexpression vector of NRF2 (pcDNA3.1-NRF2) or blank vector (pcDNA3.1) as a control, and then treated with laminarin (0.020 mg/mL). qPCR was performed to quantify the mRNA level of NRF2 (A), KEAP1 (B), NQO1 (C), GCLC (D) and HO1 (E) at 24 h post laminarin treatment. (F) Protein level of factors detected by Western blot. <t>GAPDH</t> was used as an internal control. Values with different letters indicate statistical significance ( P
    Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 87/100, based on 15555 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti keap1 antibodies
    Expression of <t>Keap1</t> Protein in the 6 experimental groups of mice. The upper bands (a) depict representative findings in the control, DIO, DEHP, DIO + DEHP, DIO + DEHP middle, and DIO + DEHP high groups. The lower bar graphs (b) show the results of the semiquantitative measurement of Keap1. Each bar represents mean ± SE. n = 4. ∗ indicates a significant difference from the control group, P
    Anti Keap1 Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc keap1
    Resisting C-2 induced apoptosis by p62 activated Nrf2 pathway in early time. a Western blotting assay was used to detect the expression levels of p62, Nrf2, <t>Keap1</t> and NQO1 after treated with 4 μM of C-2 for 24 h in BIU87 and EJ cells. b BIU87 cells were treated with C-2 for 6 h. The treated and untreated samples were stained with Nrf2 antibody (Green) and p62 antibody (Red) and DAPI (Blue) (magnification, 400X). The arrow was indicating Nrf2 nuclear translocation. c Immunoprecipitation assay showed the effect of C-2 on the binding of p62 and Keap1 proteins in BIU87 cells for 6 h and 12 h. d Western blotting assay showed the effect of p62 siRNA (20 nM) on expression of p62 and p-Nrf2 proteins in BIU87 cells. e MTT assay detected the effect of siRNA targeting to p62 on the survival rate of BIU87 cells incubated with 4 μM of C-2 for 6 h, ** P
    Keap1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti keap1
    Comparison of Cys273 and Cys288 between <t>Keap1</t> and human KLHL (Kelch-like) family members. Cys273 and 288 are highly conserved among mammal Keap1 but not in other KLHL family members. Note that whereas Cys273 of Keap1 corresponds to leucine (L) in other KLHL family members, Cys288 of Keap1 corresponds to glutamate (E) or glutamine (N) in other KLHL family members.
    Anti Keap1, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti keap1 antibody
    Structure of the interface between <t>KEAP1</t> and Nrf2 and location of Cys 434 . A bottom view ( left panel ) and a side view ( right panel ) are shown. Cys 434 is pink . Residues in KEAP1 involved in direct interaction with the ETGE motif are indicated: Arg 380 , Arg 415 , and Arg 483 in dark blue ; Ser 363 , Ser 508 , Ser 555 , and Ser 602 in orange ; and Asn 382 in light blue. Numbers of the blade structure ( 1–6 ) in KEAP1 are shown around the bottom view of the molecular model.
    Anti Keap1 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti keap1 antibody
    PQ regulates cell proliferation, cell death, and autophagy by modulating <t>Keap1/p65/Nrf2</t> signal pathway. a , b Western Blot was used to verify the efficiency of p65 overexpression and Nrf2 knock-down in 16HBE cells. c , d The proliferative ability of 16HBE cells treated with 500 μM of PQ for 12 h, 24 h, 48 h, 72 h, and 96 h was detected by CCK-8 assay. e , f The apoptosis ratio of 16HBE cells treated with 150 μM PQ for 2 h was detected by FCM. g – i The autophagy-associated proteins of 16HBE cells treated with 500 μM of PQ for 48 h were detected by Western Blot. Each assay had 3 repetitions (the data are presented as mean ± SD, “*” means statistical significance, p
    Monoclonal Anti Keap1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioss polyclonal anti keap1
    PQ regulates cell proliferation, cell death, and autophagy by modulating <t>Keap1/p65/Nrf2</t> signal pathway. a , b Western Blot was used to verify the efficiency of p65 overexpression and Nrf2 knock-down in 16HBE cells. c , d The proliferative ability of 16HBE cells treated with 500 μM of PQ for 12 h, 24 h, 48 h, 72 h, and 96 h was detected by CCK-8 assay. e , f The apoptosis ratio of 16HBE cells treated with 150 μM PQ for 2 h was detected by FCM. g – i The autophagy-associated proteins of 16HBE cells treated with 500 μM of PQ for 48 h were detected by Western Blot. Each assay had 3 repetitions (the data are presented as mean ± SD, “*” means statistical significance, p
    Polyclonal Anti Keap1, supplied by Bioss, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit anti keap1 antibody
    Liver expression of Nrf2, <t>Keap1</t> and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.
    Rabbit Anti Keap1 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 81/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti keap1 polyclonal antibody
    Liver expression of Nrf2, <t>Keap1</t> and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.
    Anti Keap1 Polyclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 82/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti keap1 e20 antibodies
    Liver expression of Nrf2, <t>Keap1</t> and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.
    Anti Keap1 E20 Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteintech anti keap1 polyclonal antibody
    Liver expression of Nrf2, <t>Keap1</t> and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.
    Anti Keap1 Polyclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 75/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti keap1 antibodies
    Agt, Nrf2, HO-1, and <t>Keap1</t> expression in Tg mouse kidneys at week 16. Immunohistochemical staining for Agt ( A ), Nrf2 ( B ), HO-1 ( C ), and Keap1 ( D ) in mouse kidneys. Magnification ×200. E : WB of Agt, Nrf2, Keap1, and HO-1 expression ( a ) and quantification of their expression ( b ) in RPTs from kidneys of WT control, Cat-Tg, Akita, and Akita Cat-Tg mice. The membranes were reblotted for β-actin. Agt, Nrf2, Keap1, and HO-1 levels were normalized by corresponding β-actin levels. Values are expressed as mean ± SEM ( n = 8). *** P
    Anti Keap1 Antibodies, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 80/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology goat polyclonal anti keap1 antibody
    Agt, Nrf2, HO-1, and <t>Keap1</t> expression in Tg mouse kidneys at week 16. Immunohistochemical staining for Agt ( A ), Nrf2 ( B ), HO-1 ( C ), and Keap1 ( D ) in mouse kidneys. Magnification ×200. E : WB of Agt, Nrf2, Keap1, and HO-1 expression ( a ) and quantification of their expression ( b ) in RPTs from kidneys of WT control, Cat-Tg, Akita, and Akita Cat-Tg mice. The membranes were reblotted for β-actin. Agt, Nrf2, Keap1, and HO-1 levels were normalized by corresponding β-actin levels. Values are expressed as mean ± SEM ( n = 8). *** P
    Goat Polyclonal Anti Keap1 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 82/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti keap1 rabbit monoclonal antibody
    Agt, Nrf2, HO-1, and <t>Keap1</t> expression in Tg mouse kidneys at week 16. Immunohistochemical staining for Agt ( A ), Nrf2 ( B ), HO-1 ( C ), and Keap1 ( D ) in mouse kidneys. Magnification ×200. E : WB of Agt, Nrf2, Keap1, and HO-1 expression ( a ) and quantification of their expression ( b ) in RPTs from kidneys of WT control, Cat-Tg, Akita, and Akita Cat-Tg mice. The membranes were reblotted for β-actin. Agt, Nrf2, Keap1, and HO-1 levels were normalized by corresponding β-actin levels. Values are expressed as mean ± SEM ( n = 8). *** P
    Anti Keap1 Rabbit Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteintech keap1
    The relationships between Nrf2 and <t>Keap1</t> expression are illustrated as scattered plots with linear regression lines. In normal esophageal mucosae, Keap1 was negatively correlated to both cytoplasmic and nuclear Nrf2, whereas there was no relationship between cytoplasmic and nuclear Nrf2. In ESCC, the relationship between Keap1 and Nrf2 in cytoplasm and nucleus disappeared, while cytoplasmic Nrf2 was positively correlated to nuclear Nrf2.
    Keap1, supplied by Proteintech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit anti keap1 polyclonal antibody
    The relationships between Nrf2 and <t>Keap1</t> expression are illustrated as scattered plots with linear regression lines. In normal esophageal mucosae, Keap1 was negatively correlated to both cytoplasmic and nuclear Nrf2, whereas there was no relationship between cytoplasmic and nuclear Nrf2. In ESCC, the relationship between Keap1 and Nrf2 in cytoplasm and nucleus disappeared, while cytoplasmic Nrf2 was positively correlated to nuclear Nrf2.
    Rabbit Anti Keap1 Polyclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology mouse anti human keap1 antibody
    The relationships between Nrf2 and <t>Keap1</t> expression are illustrated as scattered plots with linear regression lines. In normal esophageal mucosae, Keap1 was negatively correlated to both cytoplasmic and nuclear Nrf2, whereas there was no relationship between cytoplasmic and nuclear Nrf2. In ESCC, the relationship between Keap1 and Nrf2 in cytoplasm and nucleus disappeared, while cytoplasmic Nrf2 was positively correlated to nuclear Nrf2.
    Mouse Anti Human Keap1 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti rat keap1 antibody
    The relationships between Nrf2 and <t>Keap1</t> expression are illustrated as scattered plots with linear regression lines. In normal esophageal mucosae, Keap1 was negatively correlated to both cytoplasmic and nuclear Nrf2, whereas there was no relationship between cytoplasmic and nuclear Nrf2. In ESCC, the relationship between Keap1 and Nrf2 in cytoplasm and nucleus disappeared, while cytoplasmic Nrf2 was positively correlated to nuclear Nrf2.
    Rabbit Anti Rat Keap1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems human mouse rat keap1 antibody
    The relationships between Nrf2 and <t>Keap1</t> expression are illustrated as scattered plots with linear regression lines. In normal esophageal mucosae, Keap1 was negatively correlated to both cytoplasmic and nuclear Nrf2, whereas there was no relationship between cytoplasmic and nuclear Nrf2. In ESCC, the relationship between Keap1 and Nrf2 in cytoplasm and nucleus disappeared, while cytoplasmic Nrf2 was positively correlated to nuclear Nrf2.
    Human Mouse Rat Keap1 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 80/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Santa Cruz Biotechnology anti inrf2
    Cul3–Rbx1 export out of the nucleus by means of the <t>INrf2</t> nuclear export signal. ( A , B ) Immunocytochemistry: HepG2 cells (A) or Hepa-1 cells (B) were grown in Lab-Tek II chamber slides. Cells were fixed, permeabilized and incubated with a 1:500 dilution of anti-goat INrf2, anti-rabbit Cul3 and anti-rabbit Rbx1 antibody, as indicated in the figures. Cells were washed and incubated with Alexa-Fluor-594-conjugated anti-goat antibody or FITC-conjugated anti-rabbit secondary antibody (Invitrogen). Cells were washed twice with PBS, stained with Vectashield containing nuclear DAPI stain, observed under a Nikon fluorescence microscope and photographed. ( C ) Hepa-1 cells were transfected with INrf2 siRNA for 48 hours. Cells were then treated with 100 μM t -BHQ for the indicated periods of time and harvested. Cytosolic and nuclear extracts were prepared, and lysates were immunoblotted with antibodies against INrf2, Cul3, Rbx1, LDH and lamin B. ( D ) Schematic diagram of the mouse INrf2 gene showing the BTB domain (responsible for Cul3 interaction), DGR domain (responsible for Nrf2 interaction) and a functional nuclear export signal (NES). ( E ) HepG2 cells were treated and transfected with 1 μg of vector encoding INrf2ΔNES–V5. Cells were then treated with 100 μM t -BHQ, harvested and nuclear and cytosolic extracts were then prepared. Lysates were immunoblotted with antibodies against V5, Cul3, Rbx1, lamin B and LDH. ( F ) HepG2 cells were co-transfected with 1 μg of INrf2–V5 or INrf2Y85A–V5 and 1 μg of FLAG–Crm1 and then treated with either DMSO or 100 μM t -BHQ for the indicated periods of time (left and right panels). Cells were harvested; 1 mg of lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to FLAG and V5 (top two panels). 1 mg of lysate was immunoprecipitated with antibody to FLAG and western blotted with antibodies to V5 and FLAG (bottom two panels).
    Anti Inrf2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam mouse anti keap1 antibody
    Cav-1 prevents MnSOD upregulation by repression of Nrf2 A. Cav-1 (green) transfected MCF7 cells demonstrate a reduction of Nrf2 (purple) and MnSOD (red). B. H 2 O 2 production in MCF7 cells expressing Cav-1 is reduced when compared to MCF7 cells devoid of Cav-1. H 2 O 2 was measured as described in methods . C. Cysteine sulfenic acid formation in MCF7 empty vector and Cav-1 expression cells using dimedone in combination with immunoprecipitation of <t>Keap1</t> shows a reduction in Keap1 thiol oxidation in Cav-1 expressing MCF7 cells. D. Immunoprecipitation of Nrf2 (top) and Keap1 (bottom) demonstrates direct interactions of both proteins to Cav-1. Cells were treated with 400 μM H 2 O 2 for 1 h prior to preparation of lysates for immunoprecipitation as described in methods . E. Nrf-2 transcriptional activity was indirectly determined using NanoLuc Nrf2-stability luciferase reporter assay in MCF7 cells with and without Cav-1 stable expression in both DMEM:F12 and DMEM high glucose (4.5g/L) conditions. F. MnSOD mRNA expression following Cav-1 ectopic expression or silencing of Nrf2 in MCF7 cells shows a similar reduction in MnSOD mRNA. (Student's two-sided t -test, # = p = 0.06, * = p
    Mouse Anti Keap1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech anti kelch like ech associated protein 1 keap1 antibodies
    Cav-1 prevents MnSOD upregulation by repression of Nrf2 A. Cav-1 (green) transfected MCF7 cells demonstrate a reduction of Nrf2 (purple) and MnSOD (red). B. H 2 O 2 production in MCF7 cells expressing Cav-1 is reduced when compared to MCF7 cells devoid of Cav-1. H 2 O 2 was measured as described in methods . C. Cysteine sulfenic acid formation in MCF7 empty vector and Cav-1 expression cells using dimedone in combination with immunoprecipitation of <t>Keap1</t> shows a reduction in Keap1 thiol oxidation in Cav-1 expressing MCF7 cells. D. Immunoprecipitation of Nrf2 (top) and Keap1 (bottom) demonstrates direct interactions of both proteins to Cav-1. Cells were treated with 400 μM H 2 O 2 for 1 h prior to preparation of lysates for immunoprecipitation as described in methods . E. Nrf-2 transcriptional activity was indirectly determined using NanoLuc Nrf2-stability luciferase reporter assay in MCF7 cells with and without Cav-1 stable expression in both DMEM:F12 and DMEM high glucose (4.5g/L) conditions. F. MnSOD mRNA expression following Cav-1 ectopic expression or silencing of Nrf2 in MCF7 cells shows a similar reduction in MnSOD mRNA. (Student's two-sided t -test, # = p = 0.06, * = p
    Anti Kelch Like Ech Associated Protein 1 Keap1 Antibodies, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Protein expression levels of (A) Nrf2, (B) <t>Keap1</t> and (C) SOD1 were examined using western blot analysis. (D) Representative western blot analysis gel using samples from in the skeletal muscles of the control, exercise, HFD and HFD + exercise groups. Western blot analysis data are presented as mean ± standard deviation. *P
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    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

    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

    NFE2L2, KEAP1, and CUL3 mutations

    Journal: Journal of Thoracic Disease

    Article Title: Mutations and expression of the NFE2L2/KEAP1/CUL3 pathway in Chinese patients with lung squamous cell carcinoma

    doi: 10.21037/jtd.2016.06.08

    Figure Lengend Snippet: NFE2L2, KEAP1, and CUL3 mutations

    Article Snippet: Briefly, the antibodies specific for NFE2L2 (1:200 dilution, Abcam, Cambridge, UK), KEAP1 (1:200 dilution, Abcam) and CUL3 (1:200 dilution, Abcam) were used to detect these three proteins.

    Techniques:

    NFE2L2, KEAP1, and CUL3 expression

    Journal: Journal of Thoracic Disease

    Article Title: Mutations and expression of the NFE2L2/KEAP1/CUL3 pathway in Chinese patients with lung squamous cell carcinoma

    doi: 10.21037/jtd.2016.06.08

    Figure Lengend Snippet: NFE2L2, KEAP1, and CUL3 expression

    Article Snippet: Briefly, the antibodies specific for NFE2L2 (1:200 dilution, Abcam, Cambridge, UK), KEAP1 (1:200 dilution, Abcam) and CUL3 (1:200 dilution, Abcam) were used to detect these three proteins.

    Techniques: Expressing

    Expression of the NFE2L2/KEAP1/CUL3 pathway by TaqMan RT-qPCR. NFE2L2, nuclear factor erythroid 2-like 2; KEAP1, kelch-like ECH-associated protein 1; CUL3, cullin 3.

    Journal: Journal of Thoracic Disease

    Article Title: Mutations and expression of the NFE2L2/KEAP1/CUL3 pathway in Chinese patients with lung squamous cell carcinoma

    doi: 10.21037/jtd.2016.06.08

    Figure Lengend Snippet: Expression of the NFE2L2/KEAP1/CUL3 pathway by TaqMan RT-qPCR. NFE2L2, nuclear factor erythroid 2-like 2; KEAP1, kelch-like ECH-associated protein 1; CUL3, cullin 3.

    Article Snippet: Briefly, the antibodies specific for NFE2L2 (1:200 dilution, Abcam, Cambridge, UK), KEAP1 (1:200 dilution, Abcam) and CUL3 (1:200 dilution, Abcam) were used to detect these three proteins.

    Techniques: Expressing, Quantitative RT-PCR

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

    Journal: Journal of neurochemistry

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

    doi: 10.1111/jnc.13837

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

    Article Snippet: Blots were then incubated overnight at 4°C with antibodies against Nrf2 (polyclonal; 1:4,000; Thermo-Fisher Scientific; Waltham, MA), Keap1 (polyclonal; 1:2,000; Abcam; Cambridge, MA), cullin-3 (monoclonal; 1:2000; Sigma), xCT (polyclonal; 1:2000; Abcam), GSS (polyclonal; 1:1,000; Abcam), γ-GT (monoclonal; 1:2,000; Abcam), GCLc (polyclonal; 1:500; Abcam), GAPDH (monoclonal; 1:2,000; Santa Cruz Biotechnology; Santa Cruz, CA), GSK-3β (monoclonal; 1:2000; Cell Signaling; Danvers, MA), phospho-GSK-3β (S9) (monoclonal; 1:2000; Cell Signaling) and phospho-GSK-3β (Y216) (polyclonal; 1:2000; Abcam).

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

    NRF2 regulates KEAP1, NQO1, GCLC and HO1 in MRC-5 cells treated with laminarin. MRC-5 cells were transfected with the overexpression vector of NRF2 (pcDNA3.1-NRF2) or blank vector (pcDNA3.1) as a control, and then treated with laminarin (0.020 mg/mL). qPCR was performed to quantify the mRNA level of NRF2 (A), KEAP1 (B), NQO1 (C), GCLC (D) and HO1 (E) at 24 h post laminarin treatment. (F) Protein level of factors detected by Western blot. GAPDH was used as an internal control. Values with different letters indicate statistical significance ( P

    Journal: PeerJ

    Article Title: Laminarin protects against hydrogen peroxide-induced oxidative damage in MRC-5 cells possibly via regulating NRF2

    doi: 10.7717/peerj.3642

    Figure Lengend Snippet: NRF2 regulates KEAP1, NQO1, GCLC and HO1 in MRC-5 cells treated with laminarin. MRC-5 cells were transfected with the overexpression vector of NRF2 (pcDNA3.1-NRF2) or blank vector (pcDNA3.1) as a control, and then treated with laminarin (0.020 mg/mL). qPCR was performed to quantify the mRNA level of NRF2 (A), KEAP1 (B), NQO1 (C), GCLC (D) and HO1 (E) at 24 h post laminarin treatment. (F) Protein level of factors detected by Western blot. GAPDH was used as an internal control. Values with different letters indicate statistical significance ( P

    Article Snippet: The membranes were incubated in Tris-buffered saline and Tween 20 (TBST) containing the specific primary antibodies against NRF2, KEAP1, NQO1, GCLC, HO1 and GAPDH (ab89443, ab150654, ab28947, ab55435, ab13248 and ab8245; Abcam, Cambridge, UK) overnight at 4 °C.

    Techniques: Transfection, Over Expression, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot

    Nuclear factor erythroid 2 like 2 (NRF2) regulates oxidative damage in MRC-5 cells. NRF2 was knocked down by its specific siRNA (siNRF2), and cells were treated with hydrogen peroxide ( H 2 O 2 , 600 µM) and laminarin (0.020 mg/mL). Assays were performed at 24 h post treatment. (A) NRF2 mRNA level quantified by qPCR. (B) NRF2 protein level detected by Western blot. GAPDH was used as an internal reference. (C) Cellular concentration of superoxide dismutase (SOD), malondialdehyde (MDA), deduced glutathione (GSH) and catalase (CAT) quantified by biochemical assays. Values with different letters indicate statistical significance ( P

    Journal: PeerJ

    Article Title: Laminarin protects against hydrogen peroxide-induced oxidative damage in MRC-5 cells possibly via regulating NRF2

    doi: 10.7717/peerj.3642

    Figure Lengend Snippet: Nuclear factor erythroid 2 like 2 (NRF2) regulates oxidative damage in MRC-5 cells. NRF2 was knocked down by its specific siRNA (siNRF2), and cells were treated with hydrogen peroxide ( H 2 O 2 , 600 µM) and laminarin (0.020 mg/mL). Assays were performed at 24 h post treatment. (A) NRF2 mRNA level quantified by qPCR. (B) NRF2 protein level detected by Western blot. GAPDH was used as an internal reference. (C) Cellular concentration of superoxide dismutase (SOD), malondialdehyde (MDA), deduced glutathione (GSH) and catalase (CAT) quantified by biochemical assays. Values with different letters indicate statistical significance ( P

    Article Snippet: The membranes were incubated in Tris-buffered saline and Tween 20 (TBST) containing the specific primary antibodies against NRF2, KEAP1, NQO1, GCLC, HO1 and GAPDH (ab89443, ab150654, ab28947, ab55435, ab13248 and ab8245; Abcam, Cambridge, UK) overnight at 4 °C.

    Techniques: Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay, Multiple Displacement Amplification

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

    Journal: Oxidative Medicine and Cellular Longevity

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

    doi: 10.1155/2018/1861984

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

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

    Techniques: Expressing, Mouse Assay

    Resisting C-2 induced apoptosis by p62 activated Nrf2 pathway in early time. a Western blotting assay was used to detect the expression levels of p62, Nrf2, Keap1 and NQO1 after treated with 4 μM of C-2 for 24 h in BIU87 and EJ cells. b BIU87 cells were treated with C-2 for 6 h. The treated and untreated samples were stained with Nrf2 antibody (Green) and p62 antibody (Red) and DAPI (Blue) (magnification, 400X). The arrow was indicating Nrf2 nuclear translocation. c Immunoprecipitation assay showed the effect of C-2 on the binding of p62 and Keap1 proteins in BIU87 cells for 6 h and 12 h. d Western blotting assay showed the effect of p62 siRNA (20 nM) on expression of p62 and p-Nrf2 proteins in BIU87 cells. e MTT assay detected the effect of siRNA targeting to p62 on the survival rate of BIU87 cells incubated with 4 μM of C-2 for 6 h, ** P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: SP600125 enhances C-2-induced cell death by the switch from autophagy to apoptosis in bladder cancer cells

    doi: 10.1186/s13046-019-1467-6

    Figure Lengend Snippet: Resisting C-2 induced apoptosis by p62 activated Nrf2 pathway in early time. a Western blotting assay was used to detect the expression levels of p62, Nrf2, Keap1 and NQO1 after treated with 4 μM of C-2 for 24 h in BIU87 and EJ cells. b BIU87 cells were treated with C-2 for 6 h. The treated and untreated samples were stained with Nrf2 antibody (Green) and p62 antibody (Red) and DAPI (Blue) (magnification, 400X). The arrow was indicating Nrf2 nuclear translocation. c Immunoprecipitation assay showed the effect of C-2 on the binding of p62 and Keap1 proteins in BIU87 cells for 6 h and 12 h. d Western blotting assay showed the effect of p62 siRNA (20 nM) on expression of p62 and p-Nrf2 proteins in BIU87 cells. e MTT assay detected the effect of siRNA targeting to p62 on the survival rate of BIU87 cells incubated with 4 μM of C-2 for 6 h, ** P

    Article Snippet: SAPK/JNK siRNA (#6232), p62 siRNA (#6394), Poly (ADP-ribose) polymerase-1 (PARP-1) (#9532), Cleaved PARP (#5625), NQO1 (#3187), Keap1 (#4617), Bcl-xL (#2764), XIAP (#14334), JNK (#9252), p-JNK (#9255), LC3B (#3868S) and Autophagy antibody sampler kit (#4445) including Beclin-1, ATG16, ATG3, ATG7 and ATG5 were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Western Blot, Expressing, Staining, Translocation Assay, Immunoprecipitation, Binding Assay, MTT Assay, Incubation

    Effects of Nrf2 knockdown on cisplatin sensitivity in resistant A2780cp cells. A. After cells were transfected with Nrf2 siRNA (100 nM) for 48 h, cell lysates were collected for western blot analyses for Nrf2, Keap1, NQO1 and HO-1. B. Cells were transfected

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Nrf2 induces cisplatin resistance through activation of autophagy in ovarian carcinoma

    doi:

    Figure Lengend Snippet: Effects of Nrf2 knockdown on cisplatin sensitivity in resistant A2780cp cells. A. After cells were transfected with Nrf2 siRNA (100 nM) for 48 h, cell lysates were collected for western blot analyses for Nrf2, Keap1, NQO1 and HO-1. B. Cells were transfected

    Article Snippet: The membranes were incubated with rabbit monoclonal antibodies for Atg3, Atg5, beclin 1, Atg12 and Keap1 (1:1000) from Cell Signaling Technology; rabbit monoclonal antibody Nrf2 (1:20000) from Abcam; mouse monoclonal antibody NQO1 (1:2000) from Santa Cruz Biotechnology; rabbit polyclonal antibody p62 (1:500) from Proteintech; rabbit polyclonal antibody HO-1 (1:1000) from Enzo Life Science.

    Techniques: Transfection, Western Blot

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

    Journal: The Journal of Experimental Medicine

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

    doi: 10.1084/jem.20151208

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

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

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

    The effect of H 2 S on Keap1 s-sulfhydration in renal tissues of Dahl rats and SD rats. (a) Keap1 s-sulfhydration in renal tissues of Dahl rats. (b) Keap1 s-sulfhydration in renal tissues of SD rats (mean ± SE, n = 10). ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Hydrogen Sulfide Inhibits High-Salt Diet-Induced Renal Oxidative Stress and Kidney Injury in Dahl Rats

    doi: 10.1155/2016/2807490

    Figure Lengend Snippet: The effect of H 2 S on Keap1 s-sulfhydration in renal tissues of Dahl rats and SD rats. (a) Keap1 s-sulfhydration in renal tissues of Dahl rats. (b) Keap1 s-sulfhydration in renal tissues of SD rats (mean ± SE, n = 10). ∗ P

    Article Snippet: The proteins were separated in 10% SDS-PAGE and detected with Nrf2 (Enzolife, USA) or Keap1 antibody (Cell Signaling Technology, USA) by Western blotting.

    Techniques:

    The effect of H 2 S on the association of Keap1 with Nrf2 in renal tissues of Dahl rats and SD rats. (a) The association of Keap1 with Nrf2 in renal tissues of Dahl rats. (b) The association of Keap1 with Nrf2 in renal tissues of SD rats (mean ± SE, n = 10). ∗∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Hydrogen Sulfide Inhibits High-Salt Diet-Induced Renal Oxidative Stress and Kidney Injury in Dahl Rats

    doi: 10.1155/2016/2807490

    Figure Lengend Snippet: The effect of H 2 S on the association of Keap1 with Nrf2 in renal tissues of Dahl rats and SD rats. (a) The association of Keap1 with Nrf2 in renal tissues of Dahl rats. (b) The association of Keap1 with Nrf2 in renal tissues of SD rats (mean ± SE, n = 10). ∗∗ P

    Article Snippet: The proteins were separated in 10% SDS-PAGE and detected with Nrf2 (Enzolife, USA) or Keap1 antibody (Cell Signaling Technology, USA) by Western blotting.

    Techniques:

    AR inhibition augments HG- induced Nrf2 activation in Thp1 cells. Thp1 cells were treated with fidarestat (10 μ M) for indicated times. Subsequently, the cells were also pretreated with fidarestat for overnight followed by incubation with HG for 30, 60, 120, and 240 minutes. Equal amounts of nuclear and cytosolic proteins were subjected to Western blot analysis for the expression of Nrf2 and Keap1, respectively. Histone H3 and GAPDH served as loading controls for nuclear and cytosolic protein extract, respectively. A representative blot from three independent analyses is shown (a). Nrf2 transcription factor assay using the nuclear protein of treated Thp1 cells was carried out using an ELISA kit (b). Data represent mean ± SD ( n = 5). ∗ p

    Journal: Journal of Diabetes Research

    Article Title: Aldose Reductase Inhibitor Protects against Hyperglycemic Stress by Activating Nrf2-Dependent Antioxidant Proteins

    doi: 10.1155/2017/6785852

    Figure Lengend Snippet: AR inhibition augments HG- induced Nrf2 activation in Thp1 cells. Thp1 cells were treated with fidarestat (10 μ M) for indicated times. Subsequently, the cells were also pretreated with fidarestat for overnight followed by incubation with HG for 30, 60, 120, and 240 minutes. Equal amounts of nuclear and cytosolic proteins were subjected to Western blot analysis for the expression of Nrf2 and Keap1, respectively. Histone H3 and GAPDH served as loading controls for nuclear and cytosolic protein extract, respectively. A representative blot from three independent analyses is shown (a). Nrf2 transcription factor assay using the nuclear protein of treated Thp1 cells was carried out using an ELISA kit (b). Data represent mean ± SD ( n = 5). ∗ p

    Article Snippet: Antibodies against KEAP1, HO1, NQO1, phospho-AMPK-α 1, AMPK-α 1, GAPDH, and histone H3 were purchased from Cell Signal Inc. Fidarestat was obtained from Livwel Therapeutics Inc., USA.

    Techniques: Inhibition, Activation Assay, Incubation, Western Blot, Expressing, Transcription Factor Assay, Enzyme-linked Immunosorbent Assay

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.00868-15

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

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

    Techniques:

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.00868-15

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

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

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

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.00868-15

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

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

    Techniques: Dissection, Western Blot

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.00868-15

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

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

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

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.00868-15

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

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

    Techniques:

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.00868-15

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

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

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

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.00868-15

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

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

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

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.00868-15

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

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

    Techniques: Mutagenesis, Western Blot

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.00868-15

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

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

    Techniques: Functional Assay, Western Blot, Expressing

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.00868-15

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

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

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

    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

    Structure of the interface between KEAP1 and Nrf2 and location of Cys 434 . A bottom view ( left panel ) and a side view ( right panel ) are shown. Cys 434 is pink . Residues in KEAP1 involved in direct interaction with the ETGE motif are indicated: Arg 380 , Arg 415 , and Arg 483 in dark blue ; Ser 363 , Ser 508 , Ser 555 , and Ser 602 in orange ; and Asn 382 in light blue. Numbers of the blade structure ( 1–6 ) in KEAP1 are shown around the bottom view of the molecular model.

    Journal: The Journal of Biological Chemistry

    Article Title: The Critical Role of Nitric Oxide Signaling, via Protein S-Guanylation and Nitrated Cyclic GMP, in the Antioxidant Adaptive Response *

    doi: 10.1074/jbc.M110.145441

    Figure Lengend Snippet: Structure of the interface between KEAP1 and Nrf2 and location of Cys 434 . A bottom view ( left panel ) and a side view ( right panel ) are shown. Cys 434 is pink . Residues in KEAP1 involved in direct interaction with the ETGE motif are indicated: Arg 380 , Arg 415 , and Arg 483 in dark blue ; Ser 363 , Ser 508 , Ser 555 , and Ser 602 in orange ; and Asn 382 in light blue. Numbers of the blade structure ( 1–6 ) in KEAP1 are shown around the bottom view of the molecular model.

    Article Snippet: Other antibodies used in the Western blotting analysis were as follows: anti-Keap1 antibody (rat monoclonal) , anti-actin antibody (C-11, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), anti-iNOS antibody (Santa Cruz Biotechnology, Inc.), anti-FLAG M2 antibody (Sigma), anti-HO-1 antibody (Stressgen Bioreagents, Victoria, Canada), and anti-NQO1 (NAD(P)H dehydrogenase, quinone 1) antibody (Santa Cruz Biotechnology, Inc.).

    Techniques:

    Protein S -guanylation in C6 cells treated with LPS plus cytokines. A , cells were stimulated with a mixture of LPS (10 μg/ml), IFN-γ (100 units/ml), TNFα (100 units/ml), and IL-1β (10 ng/ml). Cell lysates (10 μg of protein) were analyzed by using Western blotting with anti- S -guanylated protein (8-RS-cGMP) antibody ( left ) and anti-Keap1 antibody ( right ). The 70-kDa protein ( arrowhead in the left panel ), detectable only after treatment with LPS plus cytokines, showed electrophoretic mobility identical to that of Keap1 ( arrowhead in the right panel ). B , cells without or with transfection with Keap1 siRNA were stimulated with LPS plus cytokines as in A . Protein S -guanylation and Keap1 expression were analyzed by Western blotting.

    Journal: The Journal of Biological Chemistry

    Article Title: The Critical Role of Nitric Oxide Signaling, via Protein S-Guanylation and Nitrated Cyclic GMP, in the Antioxidant Adaptive Response *

    doi: 10.1074/jbc.M110.145441

    Figure Lengend Snippet: Protein S -guanylation in C6 cells treated with LPS plus cytokines. A , cells were stimulated with a mixture of LPS (10 μg/ml), IFN-γ (100 units/ml), TNFα (100 units/ml), and IL-1β (10 ng/ml). Cell lysates (10 μg of protein) were analyzed by using Western blotting with anti- S -guanylated protein (8-RS-cGMP) antibody ( left ) and anti-Keap1 antibody ( right ). The 70-kDa protein ( arrowhead in the left panel ), detectable only after treatment with LPS plus cytokines, showed electrophoretic mobility identical to that of Keap1 ( arrowhead in the right panel ). B , cells without or with transfection with Keap1 siRNA were stimulated with LPS plus cytokines as in A . Protein S -guanylation and Keap1 expression were analyzed by Western blotting.

    Article Snippet: Other antibodies used in the Western blotting analysis were as follows: anti-Keap1 antibody (rat monoclonal) , anti-actin antibody (C-11, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), anti-iNOS antibody (Santa Cruz Biotechnology, Inc.), anti-FLAG M2 antibody (Sigma), anti-HO-1 antibody (Stressgen Bioreagents, Victoria, Canada), and anti-NQO1 (NAD(P)H dehydrogenase, quinone 1) antibody (Santa Cruz Biotechnology, Inc.).

    Techniques: Western Blot, Transfection, Expressing

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

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

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

    doi: 10.1155/2015/187265

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

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

    Techniques: Expressing

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

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

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

    doi: 10.1155/2015/187265

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

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

    Techniques: Expressing

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

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

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

    doi: 10.1155/2015/187265

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

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

    Techniques:

    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

    miR-125B antagomiR and miR-29B mimic gene targets in AML cells. ( a ) THP-1 were transfected with control miRNA, miR-125B antagomiR ( α 125B), miR-29B mimic (29B mimic) and miR-125B antagomiR in combination with miR-29B mimic for 48 h before cells were analysed for target gene expression using QRT-PCR. ( b ) THP-1 cells were transfected with control miRNA, miR-125B antagomiR ( α 125B), miR-29B mimic (29B mimic) and miR-125B antagomiR in combination with miR-29B mimic for 48 h before cells were analysed for AKT2, STAT3 and BAK1 using western blotting. Blots were reprobed for β -actin to show sample loading. ( c ) QRT-PCR of mRNA for AKT2, STAT3, BAK1 and MCL1 in THP-1 cells transduced with NEG-KD, NRF2-KD or KEAP1-KD. Values represent fold change in RNA expression over NEG-KD control. ( d ) THP-1 cells were transduced with NEG-KD, NRF2-KD or KEAP1-KD before cells were analysed for AKT2, STAT3 and BAK1 protein expression using western blotting. Blots were reprobed for β- actin to show sample loading. The numbers under the blots indicate densitometry analysis of the blots using Image J software, and the results are expressed as fold change relative to the negative control

    Journal: Cell Death and Differentiation

    Article Title: NRF2-driven miR-125B1 and miR-29B1 transcriptional regulation controls a novel anti-apoptotic miRNA regulatory network for AML survival

    doi: 10.1038/cdd.2014.152

    Figure Lengend Snippet: miR-125B antagomiR and miR-29B mimic gene targets in AML cells. ( a ) THP-1 were transfected with control miRNA, miR-125B antagomiR ( α 125B), miR-29B mimic (29B mimic) and miR-125B antagomiR in combination with miR-29B mimic for 48 h before cells were analysed for target gene expression using QRT-PCR. ( b ) THP-1 cells were transfected with control miRNA, miR-125B antagomiR ( α 125B), miR-29B mimic (29B mimic) and miR-125B antagomiR in combination with miR-29B mimic for 48 h before cells were analysed for AKT2, STAT3 and BAK1 using western blotting. Blots were reprobed for β -actin to show sample loading. ( c ) QRT-PCR of mRNA for AKT2, STAT3, BAK1 and MCL1 in THP-1 cells transduced with NEG-KD, NRF2-KD or KEAP1-KD. Values represent fold change in RNA expression over NEG-KD control. ( d ) THP-1 cells were transduced with NEG-KD, NRF2-KD or KEAP1-KD before cells were analysed for AKT2, STAT3 and BAK1 protein expression using western blotting. Blots were reprobed for β- actin to show sample loading. The numbers under the blots indicate densitometry analysis of the blots using Image J software, and the results are expressed as fold change relative to the negative control

    Article Snippet: NRF2 and KEAP1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Transduction, RNA Expression, Software, Negative Control

    miRNA profiling of AML cells in response lentiviral NRF2 knockdown. ( a ) THP-1 cells were transduced with NEG (NEG-KD)- and NRF2 (NRF2-KD)-targeted miRNA lentiviral constructs. QRT-PCR analysis of 92 cancer-associated miRNAs in NRF2-KD THP-1 cells. Values represent change in qRT-PCR cycle threshold normalised to RNU6B (ΔCT). Dashed line indicates no change in expression. A red circle indicates miR-125B, miR221, miR-223, miR222, miR29B and miR154. ( b ) QRT-PCR of miR-125B, miR221, miR-223, miR222, miR29B, miR154, NRF2, KEAP1 and HO1 in THP-1 cells transduced with NEG-KD, NRF2-KD or KEAP1-KD. Values represent fold change in RNA expression over NEG-KD control. ( c ) THP-1 were transduced with NEG-KD, NRF2-KD or KEAP1-KD before cells were analysed for NRF2 and KEAP1 using western blotting. Blots were reprobed for β -actin to show sample loading. The numbers under the blots indicate densitometry analysis of the blots using Image J software, and the results are expressed as fold change relative to the NEG-KD control

    Journal: Cell Death and Differentiation

    Article Title: NRF2-driven miR-125B1 and miR-29B1 transcriptional regulation controls a novel anti-apoptotic miRNA regulatory network for AML survival

    doi: 10.1038/cdd.2014.152

    Figure Lengend Snippet: miRNA profiling of AML cells in response lentiviral NRF2 knockdown. ( a ) THP-1 cells were transduced with NEG (NEG-KD)- and NRF2 (NRF2-KD)-targeted miRNA lentiviral constructs. QRT-PCR analysis of 92 cancer-associated miRNAs in NRF2-KD THP-1 cells. Values represent change in qRT-PCR cycle threshold normalised to RNU6B (ΔCT). Dashed line indicates no change in expression. A red circle indicates miR-125B, miR221, miR-223, miR222, miR29B and miR154. ( b ) QRT-PCR of miR-125B, miR221, miR-223, miR222, miR29B, miR154, NRF2, KEAP1 and HO1 in THP-1 cells transduced with NEG-KD, NRF2-KD or KEAP1-KD. Values represent fold change in RNA expression over NEG-KD control. ( c ) THP-1 were transduced with NEG-KD, NRF2-KD or KEAP1-KD before cells were analysed for NRF2 and KEAP1 using western blotting. Blots were reprobed for β -actin to show sample loading. The numbers under the blots indicate densitometry analysis of the blots using Image J software, and the results are expressed as fold change relative to the NEG-KD control

    Article Snippet: NRF2 and KEAP1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Transduction, Construct, Quantitative RT-PCR, Expressing, RNA Expression, Western Blot, Software

    ERK5 controls Keap1 mRNA expression. A) 10 7 Jurkat-TAg cells were transfected with 5 μg of the empty pcDNA vector, ERK5 or a pSUPER Neo vector containing a small hairpin RNA for ERK5 (shERK5). Forty-eight hours later mRNA expression was analyzed by qPCR and presented as the % of mRNA compared to cells transfected with the control vector. B) Protein expression of cells transfected in (A). C) 10 7 Jurkat-TAg cells were transfected with 5 μg of the empty pSUPER Neo vector or with the vector encoding for the shERK5. Protein expression was analyzed by WB at different times after transfection. The data represent means ± SD; *p

    Journal: EBioMedicine

    Article Title: Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS) Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS) Production

    doi: 10.1016/j.ebiom.2015.11.045

    Figure Lengend Snippet: ERK5 controls Keap1 mRNA expression. A) 10 7 Jurkat-TAg cells were transfected with 5 μg of the empty pcDNA vector, ERK5 or a pSUPER Neo vector containing a small hairpin RNA for ERK5 (shERK5). Forty-eight hours later mRNA expression was analyzed by qPCR and presented as the % of mRNA compared to cells transfected with the control vector. B) Protein expression of cells transfected in (A). C) 10 7 Jurkat-TAg cells were transfected with 5 μg of the empty pSUPER Neo vector or with the vector encoding for the shERK5. Protein expression was analyzed by WB at different times after transfection. The data represent means ± SD; *p

    Article Snippet: The antibody against KEAP1 and MEF2 (E-17) were from Santa Cruz Biotechnology.

    Techniques: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot

    Cells performing OXPHOS activate an antioxidant response. A) Different cell lines were grown in OXPHOS medium for at least 1 month before mRNA extraction. mRNA expression was quantified by qPCR and represented as the % of mRNA compared to control cells. B) Cells were treated with 20 mM DCA for 24 and 48 h and KEAP1 and NQO1 mRNA levels were quantified by qPCR. C) The expression of different proteins was analyzed in cells growing in OXPHOS medium or treated with DCA as described above. The data represent means ± SD; *p

    Journal: EBioMedicine

    Article Title: Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS) Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS) Production

    doi: 10.1016/j.ebiom.2015.11.045

    Figure Lengend Snippet: Cells performing OXPHOS activate an antioxidant response. A) Different cell lines were grown in OXPHOS medium for at least 1 month before mRNA extraction. mRNA expression was quantified by qPCR and represented as the % of mRNA compared to control cells. B) Cells were treated with 20 mM DCA for 24 and 48 h and KEAP1 and NQO1 mRNA levels were quantified by qPCR. C) The expression of different proteins was analyzed in cells growing in OXPHOS medium or treated with DCA as described above. The data represent means ± SD; *p

    Article Snippet: The antibody against KEAP1 and MEF2 (E-17) were from Santa Cruz Biotechnology.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Increase in ROS levels is not essential for KEAP1 downregulation. A) Jurkat cells were treated with increasing concentrations of H 2 O 2 for 1 h and mRNA expression was analyzed. B) OCI-AML3 cells (left) or primary tumor cells from a BCL patient (right) were treated with 1.5 mM NAC 1 h before adding DCA (20 mM) for 24 h. Cells were labeled with CH-H2DCFDA and analyzed by FACs for ROS production. Keap1 mRNA and protein were analyzed as described in Fig. 2 . C) Primary tumor cells from 2 BCL patients were treated as in (B) before analyzing KEAP1 mRNA expression, results represent the means ± SD of these two patients in triplicate. The data represent means ± SD; *p

    Journal: EBioMedicine

    Article Title: Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS) Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS) Production

    doi: 10.1016/j.ebiom.2015.11.045

    Figure Lengend Snippet: Increase in ROS levels is not essential for KEAP1 downregulation. A) Jurkat cells were treated with increasing concentrations of H 2 O 2 for 1 h and mRNA expression was analyzed. B) OCI-AML3 cells (left) or primary tumor cells from a BCL patient (right) were treated with 1.5 mM NAC 1 h before adding DCA (20 mM) for 24 h. Cells were labeled with CH-H2DCFDA and analyzed by FACs for ROS production. Keap1 mRNA and protein were analyzed as described in Fig. 2 . C) Primary tumor cells from 2 BCL patients were treated as in (B) before analyzing KEAP1 mRNA expression, results represent the means ± SD of these two patients in triplicate. The data represent means ± SD; *p

    Article Snippet: The antibody against KEAP1 and MEF2 (E-17) were from Santa Cruz Biotechnology.

    Techniques: Expressing, Labeling, FACS

    miR-23a targets KEAP1 mRNA. A) Jurkat cells were transfected with the whole miR-23a–27a–24-2 locus or with the constructs miR-23 ∆ 24–27 and miR-23 ∆ 23. The expression of KEAP1 mRNA was analyzed by qPCR and represented as the % of mRNA compared to cells transfected with the control vector. B) Expression of KEAP1 protein and the quantification. C) Jurkat cells were transfected with the different constructs together with a reporter plasmid containing the 3′UTR of KEAP1 mRNA downstream of the luciferase mRNA. Data are represented as the % of luciferase expression in cells transfected with the empty vector. D) The expression of NQO-1 mRNA was analyzed by qPCR in cells transfected as in (A). The data represent means ± SD; *p

    Journal: EBioMedicine

    Article Title: Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS) Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS) Production

    doi: 10.1016/j.ebiom.2015.11.045

    Figure Lengend Snippet: miR-23a targets KEAP1 mRNA. A) Jurkat cells were transfected with the whole miR-23a–27a–24-2 locus or with the constructs miR-23 ∆ 24–27 and miR-23 ∆ 23. The expression of KEAP1 mRNA was analyzed by qPCR and represented as the % of mRNA compared to cells transfected with the control vector. B) Expression of KEAP1 protein and the quantification. C) Jurkat cells were transfected with the different constructs together with a reporter plasmid containing the 3′UTR of KEAP1 mRNA downstream of the luciferase mRNA. Data are represented as the % of luciferase expression in cells transfected with the empty vector. D) The expression of NQO-1 mRNA was analyzed by qPCR in cells transfected as in (A). The data represent means ± SD; *p

    Article Snippet: The antibody against KEAP1 and MEF2 (E-17) were from Santa Cruz Biotechnology.

    Techniques: Transfection, Construct, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Luciferase

    miR-101 upregulates Nrf2-dependent HO-1 expression by targeting Cul3. HUVECs were transfected with pSilencer 2.1-U6/pre-miR-101 (mir-101), pSilencer 2.1-U6/control pre-miR (C-mir), or p3xFLAG-CMV10-Cul3 or in combination with the psiCHECK™-2 vector containing wild-type or mutant 3′UTRs of Cul3, followed by incubation in fresh media for 12 h. (A) Cul3 3′UTR activity was determined by dual-luciferase report assay. (B) Cul3 and HO-1 expression levels were determined by RT-PCR and Western blotting. (C) After cells were treated with 5 μM MG132 for 4 h, ubiquitinated Nrf2 was determined by Western blotting after immunoprecipitation. (D) Cells were treated with 0.5 μg/ml actinomycin D for the indicated time period. Nrf2 protein levels were determined by Western blot analysis. Data shown are average values from two individual experiments. (E) Cell lysates were immunoprecipitated with antibodies for Keap1. Interaction between Keap1 and Nrf2 was determined by Western blotting. (F) Nrf2 nuclear translocation was determined in intact cells by immunohistochemistry. Scale bars: 25 μm. (G) Specific binding of Nrf2 to the antioxidant response element of HO-1 promoter was determined by ChIP assay. (H) HO-1 promoter activity was determined using a luciferase-base assay system. The data shown in bar graphs are the mean±SD ( n =3). * p

    Journal: Antioxidants & Redox Signaling

    Article Title: Hypoxia-Responsive MicroRNA-101 Promotes Angiogenesis via Heme Oxygenase-1/Vascular Endothelial Growth Factor Axis by Targeting Cullin 3

    doi: 10.1089/ars.2014.5856

    Figure Lengend Snippet: miR-101 upregulates Nrf2-dependent HO-1 expression by targeting Cul3. HUVECs were transfected with pSilencer 2.1-U6/pre-miR-101 (mir-101), pSilencer 2.1-U6/control pre-miR (C-mir), or p3xFLAG-CMV10-Cul3 or in combination with the psiCHECK™-2 vector containing wild-type or mutant 3′UTRs of Cul3, followed by incubation in fresh media for 12 h. (A) Cul3 3′UTR activity was determined by dual-luciferase report assay. (B) Cul3 and HO-1 expression levels were determined by RT-PCR and Western blotting. (C) After cells were treated with 5 μM MG132 for 4 h, ubiquitinated Nrf2 was determined by Western blotting after immunoprecipitation. (D) Cells were treated with 0.5 μg/ml actinomycin D for the indicated time period. Nrf2 protein levels were determined by Western blot analysis. Data shown are average values from two individual experiments. (E) Cell lysates were immunoprecipitated with antibodies for Keap1. Interaction between Keap1 and Nrf2 was determined by Western blotting. (F) Nrf2 nuclear translocation was determined in intact cells by immunohistochemistry. Scale bars: 25 μm. (G) Specific binding of Nrf2 to the antioxidant response element of HO-1 promoter was determined by ChIP assay. (H) HO-1 promoter activity was determined using a luciferase-base assay system. The data shown in bar graphs are the mean±SD ( n =3). * p

    Article Snippet: Cell lysates were incubated with antibodies against Keap1 (Santa Cruz Biotechnology), Nrf2, HIF-1α (Novus Biologicals), and HSP90 (Santa Cruz Biotechnology), and immune complexes were collected by centrifugation after incubation with an antibody against protein G-Sepharose (Millipore).

    Techniques: Expressing, Transfection, Plasmid Preparation, Mutagenesis, Incubation, Activity Assay, Luciferase, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Translocation Assay, Immunohistochemistry, Binding Assay, Chromatin Immunoprecipitation

    Schematic diagram demonstrating miR-101 promotion of angiogenesis . Hypoxia elevates the expression of miR-101, which binds to the 3′UTR of Cul3 mRNA. A reduction of Cul3 level activates Nrf2/HO-1 axis and then degrades heme to produces CO, biliverdin, and bilirubin. These products lead to HIF-1α stabilization and VEGF expression. There is a positive feedback circuit to amplify the Nrf2/HO-1 pathway via VEGF/eNOS/NO-dependent S -nitrosylation of Keap1. CO, carbon monoxide.

    Journal: Antioxidants & Redox Signaling

    Article Title: Hypoxia-Responsive MicroRNA-101 Promotes Angiogenesis via Heme Oxygenase-1/Vascular Endothelial Growth Factor Axis by Targeting Cullin 3

    doi: 10.1089/ars.2014.5856

    Figure Lengend Snippet: Schematic diagram demonstrating miR-101 promotion of angiogenesis . Hypoxia elevates the expression of miR-101, which binds to the 3′UTR of Cul3 mRNA. A reduction of Cul3 level activates Nrf2/HO-1 axis and then degrades heme to produces CO, biliverdin, and bilirubin. These products lead to HIF-1α stabilization and VEGF expression. There is a positive feedback circuit to amplify the Nrf2/HO-1 pathway via VEGF/eNOS/NO-dependent S -nitrosylation of Keap1. CO, carbon monoxide.

    Article Snippet: Cell lysates were incubated with antibodies against Keap1 (Santa Cruz Biotechnology), Nrf2, HIF-1α (Novus Biologicals), and HSP90 (Santa Cruz Biotechnology), and immune complexes were collected by centrifugation after incubation with an antibody against protein G-Sepharose (Millipore).

    Techniques: Expressing

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

    Journal: Inflammation

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

    doi: 10.1007/s10753-018-00956-1

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

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

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

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

    Journal: Inflammation

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

    doi: 10.1007/s10753-018-00956-1

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

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

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

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

    Journal: Inflammation

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

    doi: 10.1007/s10753-018-00956-1

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

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

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

    Liver expression of Nrf2, Keap1 and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.

    Journal: Scientific Reports

    Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis

    doi: 10.1038/srep44769

    Figure Lengend Snippet: Liver expression of Nrf2, Keap1 and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.

    Article Snippet: Then sections were probed with rabbit anti-Keap1 (Santa Cruz, #33569; 1:50 dilution), anti-Nrf2 (Cell Signaling, # 12721 S; 1:20 dilution), anti-CK19 (Santa Cruz, #33119; 1:50 dilution).

    Techniques: Expressing, Immunohistochemistry, Staining

    The hepatic expression of Keap1 in liver tissues of patients with PBC and controls. ( A ) Keap1 protein levels were determined with densitometry analyses, after normalization to GAPDH as a loading control. ( B ) Keap1 mRNA levels were estimated in patients with cirrhotic PBC, patients with early stage PBC, and controls. Results were normalized to 18sRNA. Bars indicate the mean ± SEM. ( C ) Representative immunofluorescence micrographs show liver sections from patients with PBC. (a) Nuclei are stained with DAPI (blue). (b) Immunofluorescence staining of Keap1 (green) shows its abundance in hepatocytes. (c) Arrows indicate the perinuclear and nuclear localizations of Keap1whereas arrowheads indicate cytoplasmic localization of Keap1.

    Journal: Scientific Reports

    Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis

    doi: 10.1038/srep44769

    Figure Lengend Snippet: The hepatic expression of Keap1 in liver tissues of patients with PBC and controls. ( A ) Keap1 protein levels were determined with densitometry analyses, after normalization to GAPDH as a loading control. ( B ) Keap1 mRNA levels were estimated in patients with cirrhotic PBC, patients with early stage PBC, and controls. Results were normalized to 18sRNA. Bars indicate the mean ± SEM. ( C ) Representative immunofluorescence micrographs show liver sections from patients with PBC. (a) Nuclei are stained with DAPI (blue). (b) Immunofluorescence staining of Keap1 (green) shows its abundance in hepatocytes. (c) Arrows indicate the perinuclear and nuclear localizations of Keap1whereas arrowheads indicate cytoplasmic localization of Keap1.

    Article Snippet: Then sections were probed with rabbit anti-Keap1 (Santa Cruz, #33569; 1:50 dilution), anti-Nrf2 (Cell Signaling, # 12721 S; 1:20 dilution), anti-CK19 (Santa Cruz, #33119; 1:50 dilution).

    Techniques: Expressing, Immunofluorescence, Staining

    Agt, Nrf2, HO-1, and Keap1 expression in Tg mouse kidneys at week 16. Immunohistochemical staining for Agt ( A ), Nrf2 ( B ), HO-1 ( C ), and Keap1 ( D ) in mouse kidneys. Magnification ×200. E : WB of Agt, Nrf2, Keap1, and HO-1 expression ( a ) and quantification of their expression ( b ) in RPTs from kidneys of WT control, Cat-Tg, Akita, and Akita Cat-Tg mice. The membranes were reblotted for β-actin. Agt, Nrf2, Keap1, and HO-1 levels were normalized by corresponding β-actin levels. Values are expressed as mean ± SEM ( n = 8). *** P

    Journal: Diabetes

    Article Title: Catalase Overexpression Prevents Nuclear Factor Erythroid 2–Related Factor 2 Stimulation of Renal Angiotensinogen Gene Expression, Hypertension, and Kidney Injury in Diabetic Mice

    doi: 10.2337/db13-1830

    Figure Lengend Snippet: Agt, Nrf2, HO-1, and Keap1 expression in Tg mouse kidneys at week 16. Immunohistochemical staining for Agt ( A ), Nrf2 ( B ), HO-1 ( C ), and Keap1 ( D ) in mouse kidneys. Magnification ×200. E : WB of Agt, Nrf2, Keap1, and HO-1 expression ( a ) and quantification of their expression ( b ) in RPTs from kidneys of WT control, Cat-Tg, Akita, and Akita Cat-Tg mice. The membranes were reblotted for β-actin. Agt, Nrf2, Keap1, and HO-1 levels were normalized by corresponding β-actin levels. Values are expressed as mean ± SEM ( n = 8). *** P

    Article Snippet: Anti-Nrf2 and anti-Keap1 antibodies were obtained from BD Biosciences (Mississauga, Ontario, Canada) and R & D Systems (Minneapolis, MN), respectively.

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

    The relationships between Nrf2 and Keap1 expression are illustrated as scattered plots with linear regression lines. In normal esophageal mucosae, Keap1 was negatively correlated to both cytoplasmic and nuclear Nrf2, whereas there was no relationship between cytoplasmic and nuclear Nrf2. In ESCC, the relationship between Keap1 and Nrf2 in cytoplasm and nucleus disappeared, while cytoplasmic Nrf2 was positively correlated to nuclear Nrf2.

    Journal: Thoracic Cancer

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

    doi: 10.1111/1759-7714.12640

    Figure Lengend Snippet: The relationships between Nrf2 and Keap1 expression are illustrated as scattered plots with linear regression lines. In normal esophageal mucosae, Keap1 was negatively correlated to both cytoplasmic and nuclear Nrf2, whereas there was no relationship between cytoplasmic and nuclear Nrf2. In ESCC, the relationship between Keap1 and Nrf2 in cytoplasm and nucleus disappeared, while cytoplasmic Nrf2 was positively correlated to nuclear Nrf2.

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

    Techniques: Expressing

    Kaplan–Meier curves for overall survival (OS) and progression‐free survival (PFS) according to Nrf2 expression in the nucleus and cytoplasm and Keap1 expression in esophageal squamous cell carcinoma (ESCC) samples. Positive Nrf2 nuclear expression was significantly related to short PFS and predicted prolonged OS without significance. Negative, Positive, Negative‐censored, Positive‐censored; Negative, Positive, Negative‐censored, Positive‐censored; Negative, Positive, Negative‐censored, Positive‐censored; and Negative, Positive, Negative‐censored, Positive‐censored. Low Keap1 expression was associated with slightly improved PFS, but no significance was achieved. Low, High, Low‐censored, High‐censored; Low, High, Low‐censored, High‐censored.

    Journal: Thoracic Cancer

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

    doi: 10.1111/1759-7714.12640

    Figure Lengend Snippet: Kaplan–Meier curves for overall survival (OS) and progression‐free survival (PFS) according to Nrf2 expression in the nucleus and cytoplasm and Keap1 expression in esophageal squamous cell carcinoma (ESCC) samples. Positive Nrf2 nuclear expression was significantly related to short PFS and predicted prolonged OS without significance. Negative, Positive, Negative‐censored, Positive‐censored; Negative, Positive, Negative‐censored, Positive‐censored; Negative, Positive, Negative‐censored, Positive‐censored; and Negative, Positive, Negative‐censored, Positive‐censored. Low Keap1 expression was associated with slightly improved PFS, but no significance was achieved. Low, High, Low‐censored, High‐censored; Low, High, Low‐censored, High‐censored.

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

    Techniques: Expressing

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

    Journal: Thoracic Cancer

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

    doi: 10.1111/1759-7714.12640

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

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

    Techniques: Immunohistochemistry, Staining, Expressing

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

    Journal: Thoracic Cancer

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

    doi: 10.1111/1759-7714.12640

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

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

    Techniques: Immunohistochemistry

    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

    Association between Nrf2 and Keap1 expression and NSCLC patient outcome using TMA specimens

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Nrf2 and Keap1 Abnormalities in Non-Small Cell Lung Carcinoma and Association with Clinicopathologic Features

    doi: 10.1158/1078-0432.CCR-09-3352

    Figure Lengend Snippet: Association between Nrf2 and Keap1 expression and NSCLC patient outcome using TMA specimens

    Article Snippet: An immunohistochemical analysis was performed using commercially available antibodies against Nrf2 (dilution 1:200, clone H300; Santa Cruz Biotechnologies), Keap1 (dilution 1:25; Proteintech), and NQO1 (dilution 1:1000, clone A180; Novus Biological, Littleton, CO).

    Techniques: Expressing

    A, Five-year OS and B , RFS rated by nuclear Nrf2 protein expression in all patients with NSCLC. C , Five-year OS and D, RFS rates by cytoplasmic Keap1 protein expression in patients with squamous cell carcinoma. E, Five-year OS and F , RFS rated by nuclear

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Nrf2 and Keap1 Abnormalities in Non-Small Cell Lung Carcinoma and Association with Clinicopathologic Features

    doi: 10.1158/1078-0432.CCR-09-3352

    Figure Lengend Snippet: A, Five-year OS and B , RFS rated by nuclear Nrf2 protein expression in all patients with NSCLC. C , Five-year OS and D, RFS rates by cytoplasmic Keap1 protein expression in patients with squamous cell carcinoma. E, Five-year OS and F , RFS rated by nuclear

    Article Snippet: An immunohistochemical analysis was performed using commercially available antibodies against Nrf2 (dilution 1:200, clone H300; Santa Cruz Biotechnologies), Keap1 (dilution 1:25; Proteintech), and NQO1 (dilution 1:1000, clone A180; Novus Biological, Littleton, CO).

    Techniques: Expressing

    Nrf2 and Keap1 immunohistochemical expression in NSCLC TMA and association with clinicopathologic and genetic features

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Nrf2 and Keap1 Abnormalities in Non-Small Cell Lung Carcinoma and Association with Clinicopathologic Features

    doi: 10.1158/1078-0432.CCR-09-3352

    Figure Lengend Snippet: Nrf2 and Keap1 immunohistochemical expression in NSCLC TMA and association with clinicopathologic and genetic features

    Article Snippet: An immunohistochemical analysis was performed using commercially available antibodies against Nrf2 (dilution 1:200, clone H300; Santa Cruz Biotechnologies), Keap1 (dilution 1:25; Proteintech), and NQO1 (dilution 1:1000, clone A180; Novus Biological, Littleton, CO).

    Techniques: Immunohistochemistry, Expressing

    Microphotographs showing positive ( blue arrows ) and negative nuclear Nrf2 expression; and absent, low and high cytoplasmic Keap1 expression in NSCLC tumor tissue specimens: squamous cell carcinoma ( A , C to E ), and adenocarcinoma ( B and F ) (magnification,

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Nrf2 and Keap1 Abnormalities in Non-Small Cell Lung Carcinoma and Association with Clinicopathologic Features

    doi: 10.1158/1078-0432.CCR-09-3352

    Figure Lengend Snippet: Microphotographs showing positive ( blue arrows ) and negative nuclear Nrf2 expression; and absent, low and high cytoplasmic Keap1 expression in NSCLC tumor tissue specimens: squamous cell carcinoma ( A , C to E ), and adenocarcinoma ( B and F ) (magnification,

    Article Snippet: An immunohistochemical analysis was performed using commercially available antibodies against Nrf2 (dilution 1:200, clone H300; Santa Cruz Biotechnologies), Keap1 (dilution 1:25; Proteintech), and NQO1 (dilution 1:1000, clone A180; Novus Biological, Littleton, CO).

    Techniques: Expressing

    Subtype-specific genomic differences are reflected at the protein level. Immunohistochemical analysis of protein levels for ERCC1 (A–C), KEAP1 (D–F) and SOX2 (G–I) in squamous and adenocarcinoma lung tumors. Average immunohistochemical protein expression levels for each subtype are plotted ± SEM of each group. Representative microphotograps showing tumoral cells (arrows) with higher levels of immunohistochemistry expression of nuclear ERCC1 ( B and C ), cytoplasmic KEAP1 ( E and F ) and nuclear SOX2 ( H and I ) in squamous cell carcinomas ( B, E and H ) compared to lung adenocarcinomas ( C, F, and I ). Images are of samples reflecting the average protein expression for each group (ERCC1: SqCC = ∼80, AC = ∼43; KEAP1: SqCC = ∼161, AC = ∼126; SOX2: SqCC = ∼207, AC = ∼70). Magnification 200x. * and ** = p

    Journal: PLoS ONE

    Article Title: Divergent Genomic and Epigenomic Landscapes of Lung Cancer Subtypes Underscore the Selection of Different Oncogenic Pathways during Tumor Development

    doi: 10.1371/journal.pone.0037775

    Figure Lengend Snippet: Subtype-specific genomic differences are reflected at the protein level. Immunohistochemical analysis of protein levels for ERCC1 (A–C), KEAP1 (D–F) and SOX2 (G–I) in squamous and adenocarcinoma lung tumors. Average immunohistochemical protein expression levels for each subtype are plotted ± SEM of each group. Representative microphotograps showing tumoral cells (arrows) with higher levels of immunohistochemistry expression of nuclear ERCC1 ( B and C ), cytoplasmic KEAP1 ( E and F ) and nuclear SOX2 ( H and I ) in squamous cell carcinomas ( B, E and H ) compared to lung adenocarcinomas ( C, F, and I ). Images are of samples reflecting the average protein expression for each group (ERCC1: SqCC = ∼80, AC = ∼43; KEAP1: SqCC = ∼161, AC = ∼126; SOX2: SqCC = ∼207, AC = ∼70). Magnification 200x. * and ** = p

    Article Snippet: Immunohistochemical analysis The immunohistochemical analysis was done using commercially available antibodies against KEAP1 (dilution1∶25; Proteintech, Chicago, IL), ERCC1 (dilution 1∶25; Labvision, Fremont, CA) and SOX2 (dilution 1∶50; R & D system, Minneapolis, MN).

    Techniques: Immunohistochemistry, Expressing

    Cul3–Rbx1 export out of the nucleus by means of the INrf2 nuclear export signal. ( A , B ) Immunocytochemistry: HepG2 cells (A) or Hepa-1 cells (B) were grown in Lab-Tek II chamber slides. Cells were fixed, permeabilized and incubated with a 1:500 dilution of anti-goat INrf2, anti-rabbit Cul3 and anti-rabbit Rbx1 antibody, as indicated in the figures. Cells were washed and incubated with Alexa-Fluor-594-conjugated anti-goat antibody or FITC-conjugated anti-rabbit secondary antibody (Invitrogen). Cells were washed twice with PBS, stained with Vectashield containing nuclear DAPI stain, observed under a Nikon fluorescence microscope and photographed. ( C ) Hepa-1 cells were transfected with INrf2 siRNA for 48 hours. Cells were then treated with 100 μM t -BHQ for the indicated periods of time and harvested. Cytosolic and nuclear extracts were prepared, and lysates were immunoblotted with antibodies against INrf2, Cul3, Rbx1, LDH and lamin B. ( D ) Schematic diagram of the mouse INrf2 gene showing the BTB domain (responsible for Cul3 interaction), DGR domain (responsible for Nrf2 interaction) and a functional nuclear export signal (NES). ( E ) HepG2 cells were treated and transfected with 1 μg of vector encoding INrf2ΔNES–V5. Cells were then treated with 100 μM t -BHQ, harvested and nuclear and cytosolic extracts were then prepared. Lysates were immunoblotted with antibodies against V5, Cul3, Rbx1, lamin B and LDH. ( F ) HepG2 cells were co-transfected with 1 μg of INrf2–V5 or INrf2Y85A–V5 and 1 μg of FLAG–Crm1 and then treated with either DMSO or 100 μM t -BHQ for the indicated periods of time (left and right panels). Cells were harvested; 1 mg of lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to FLAG and V5 (top two panels). 1 mg of lysate was immunoprecipitated with antibody to FLAG and western blotted with antibodies to V5 and FLAG (bottom two panels).

    Journal: Journal of Cell Science

    Article Title: Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression

    doi: 10.1242/jcs.097295

    Figure Lengend Snippet: Cul3–Rbx1 export out of the nucleus by means of the INrf2 nuclear export signal. ( A , B ) Immunocytochemistry: HepG2 cells (A) or Hepa-1 cells (B) were grown in Lab-Tek II chamber slides. Cells were fixed, permeabilized and incubated with a 1:500 dilution of anti-goat INrf2, anti-rabbit Cul3 and anti-rabbit Rbx1 antibody, as indicated in the figures. Cells were washed and incubated with Alexa-Fluor-594-conjugated anti-goat antibody or FITC-conjugated anti-rabbit secondary antibody (Invitrogen). Cells were washed twice with PBS, stained with Vectashield containing nuclear DAPI stain, observed under a Nikon fluorescence microscope and photographed. ( C ) Hepa-1 cells were transfected with INrf2 siRNA for 48 hours. Cells were then treated with 100 μM t -BHQ for the indicated periods of time and harvested. Cytosolic and nuclear extracts were prepared, and lysates were immunoblotted with antibodies against INrf2, Cul3, Rbx1, LDH and lamin B. ( D ) Schematic diagram of the mouse INrf2 gene showing the BTB domain (responsible for Cul3 interaction), DGR domain (responsible for Nrf2 interaction) and a functional nuclear export signal (NES). ( E ) HepG2 cells were treated and transfected with 1 μg of vector encoding INrf2ΔNES–V5. Cells were then treated with 100 μM t -BHQ, harvested and nuclear and cytosolic extracts were then prepared. Lysates were immunoblotted with antibodies against V5, Cul3, Rbx1, lamin B and LDH. ( F ) HepG2 cells were co-transfected with 1 μg of INrf2–V5 or INrf2Y85A–V5 and 1 μg of FLAG–Crm1 and then treated with either DMSO or 100 μM t -BHQ for the indicated periods of time (left and right panels). Cells were harvested; 1 mg of lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to FLAG and V5 (top two panels). 1 mg of lysate was immunoprecipitated with antibody to FLAG and western blotted with antibodies to V5 and FLAG (bottom two panels).

    Article Snippet: Antibodies used in this study were as follows: anti-INrf2 (1:1000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cul3 (1:1000), anti-Rbx1 (1:1000) purchased from Cell Signaling (Danvers, MA), anti-V5 HRP (1:5000), anti-FLAG HRP purchased from Invitrogen, anti-phosphotyrosine (1:1000) and anti-actin (1:5000) purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Immunocytochemistry, Incubation, Staining, Fluorescence, Microscopy, Transfection, Functional Assay, Plasmid Preparation, Immunoprecipitation, Western Blot

    Immunoprecipitation of Cul3 and Rbx1. ( A , B ) HepG2 cells were treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were then collected, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody to (A) Cul3 and (B) Rbx1 and western blotted with antibodies to phosphotyrosine and INrf2 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibody to Cul3, Rbx1 and phosphotyrosine (bottom panels). ( C , D ) HepG2 cells were transfected with 1 μg of Cul3–V5 (C, left panel), Rbx1–V5 (D, left panel), Cul3Y764A–V5 (C, right panel) or Rbx1Y106–V5 (D, right panel) and treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were harvested, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to phosphotyrosine and V5 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibody to V5 (bottom panels). Asterisks (*) indicate the Rbx1 bands.

    Journal: Journal of Cell Science

    Article Title: Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression

    doi: 10.1242/jcs.097295

    Figure Lengend Snippet: Immunoprecipitation of Cul3 and Rbx1. ( A , B ) HepG2 cells were treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were then collected, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody to (A) Cul3 and (B) Rbx1 and western blotted with antibodies to phosphotyrosine and INrf2 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibody to Cul3, Rbx1 and phosphotyrosine (bottom panels). ( C , D ) HepG2 cells were transfected with 1 μg of Cul3–V5 (C, left panel), Rbx1–V5 (D, left panel), Cul3Y764A–V5 (C, right panel) or Rbx1Y106–V5 (D, right panel) and treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were harvested, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to phosphotyrosine and V5 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibody to V5 (bottom panels). Asterisks (*) indicate the Rbx1 bands.

    Article Snippet: Antibodies used in this study were as follows: anti-INrf2 (1:1000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cul3 (1:1000), anti-Rbx1 (1:1000) purchased from Cell Signaling (Danvers, MA), anti-V5 HRP (1:5000), anti-FLAG HRP purchased from Invitrogen, anti-phosphotyrosine (1:1000) and anti-actin (1:5000) purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Immunoprecipitation, Western Blot, Transfection

    Subcellular localization of mutant forms of INrf2. ( A – D ) HepG2 cells were transfected with 1 μg of mutant plasmids encoding INrf2Y85A, INrf2Y141A, INrf2Y208A or INrf2Y255A. Cells were then treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were harvested and nuclear and cytosolic extracts were prepared. Lysates were immunoblotted with antibodies to V5, LDH, LaminB, Cul3 and Rbx1.

    Journal: Journal of Cell Science

    Article Title: Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression

    doi: 10.1242/jcs.097295

    Figure Lengend Snippet: Subcellular localization of mutant forms of INrf2. ( A – D ) HepG2 cells were transfected with 1 μg of mutant plasmids encoding INrf2Y85A, INrf2Y141A, INrf2Y208A or INrf2Y255A. Cells were then treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were harvested and nuclear and cytosolic extracts were prepared. Lysates were immunoblotted with antibodies to V5, LDH, LaminB, Cul3 and Rbx1.

    Article Snippet: Antibodies used in this study were as follows: anti-INrf2 (1:1000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cul3 (1:1000), anti-Rbx1 (1:1000) purchased from Cell Signaling (Danvers, MA), anti-V5 HRP (1:5000), anti-FLAG HRP purchased from Invitrogen, anti-phosphotyrosine (1:1000) and anti-actin (1:5000) purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Mutagenesis, Transfection

    Cytosolic accumulation and degradation of the INrf2–Cul3–Rbx1 complex. ( A – C ) HepG2 cells were pre-treated with 2 μM MG132 for 16 hours. HepG2 cells were then treated with 100 μM t -BHQ and MG132 for the indicated periods of time. Cells were harvested, and nuclear and cytosolic extracts were prepared. Lysates were immunoblotted. The densitometry measurements of bands were quantified and relative intensities are presented below the blots by numbers. (A) Endogenous INrf2, Cul3, Rbx1, LDH and LaminB were probed. (B) 1 μg of vector encoding INrf2–V5 was transiently transfected; INrf2–V5, Cul3, Rbx1, LDH and lamin B were probed. (C) 1 μg of vector encoding the INrf2Y85A–V5 mutant was transiently transfected; INrf2Y85A–V5, Cul3, Rbx1, LDH and lamin B were probed. ( D – F ) HepG2 cells were transfected with vector encoding INrf2–V5, Cul3–V5 or Myc–Rbx1 and HA–Ub and pre-treated with 2 μM MG132 for 16 hours. Cells were then treated with 100 μM t -BHQ and MG132 for the indicated periods of time. Protein was aliquoted from samples and used for inputs. The rest of the sample (1 mg) was immunoprecipitated with antibodies to V5 or myc and immunoblotted with antibodies to HA.

    Journal: Journal of Cell Science

    Article Title: Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression

    doi: 10.1242/jcs.097295

    Figure Lengend Snippet: Cytosolic accumulation and degradation of the INrf2–Cul3–Rbx1 complex. ( A – C ) HepG2 cells were pre-treated with 2 μM MG132 for 16 hours. HepG2 cells were then treated with 100 μM t -BHQ and MG132 for the indicated periods of time. Cells were harvested, and nuclear and cytosolic extracts were prepared. Lysates were immunoblotted. The densitometry measurements of bands were quantified and relative intensities are presented below the blots by numbers. (A) Endogenous INrf2, Cul3, Rbx1, LDH and LaminB were probed. (B) 1 μg of vector encoding INrf2–V5 was transiently transfected; INrf2–V5, Cul3, Rbx1, LDH and lamin B were probed. (C) 1 μg of vector encoding the INrf2Y85A–V5 mutant was transiently transfected; INrf2Y85A–V5, Cul3, Rbx1, LDH and lamin B were probed. ( D – F ) HepG2 cells were transfected with vector encoding INrf2–V5, Cul3–V5 or Myc–Rbx1 and HA–Ub and pre-treated with 2 μM MG132 for 16 hours. Cells were then treated with 100 μM t -BHQ and MG132 for the indicated periods of time. Protein was aliquoted from samples and used for inputs. The rest of the sample (1 mg) was immunoprecipitated with antibodies to V5 or myc and immunoblotted with antibodies to HA.

    Article Snippet: Antibodies used in this study were as follows: anti-INrf2 (1:1000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cul3 (1:1000), anti-Rbx1 (1:1000) purchased from Cell Signaling (Danvers, MA), anti-V5 HRP (1:5000), anti-FLAG HRP purchased from Invitrogen, anti-phosphotyrosine (1:1000) and anti-actin (1:5000) purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Plasmid Preparation, Transfection, Mutagenesis, Immunoprecipitation

    Nuclear accumulation of INrf2Y85A shows no effect on Nrf2 activation but interferes with nuclear removal of Nrf2. ( A ) Nrf2 ubiquitylation. HepG2 cells were co-transfected with constructs encoding FLAG–Nrf2, INrf2–V5 or mutant INrf2Y85A–V5 and HA–Ub for 24 hours. Cells were pretreated with MG132 (5 μM) for 2 hours and post-treated with DMSO or t -BHQ (100 μM) for the indicated periods of time. Cells were harvested and nuclear and cytosolic extracts prepared by Active Motif Kit. 500 μg of nuclear and cytosolic extracts were immunoprecipitated with antibody against FLAG and western blotted for HA–HRP antibody (left top panels). Nuclear and cytosolic ubiquitylation of FLAG–Nrf2 was measured by using Quantity-One 4.6.3 Image software (ChemiDoc XRS, Bio-Rad) and plotted (right top panels). 60 μg of nuclear lysates from the above were also immunoblotted with antibodies against FLAG, V5 and lamin-B (left bottom panel). After immunoblotting, the intensities of protein bands of nuclear FLAG–Nrf2, INrf2–V5 and mutant INrf2Y85A–V5 were measured by using Quantity-One 4.6.3. Image software, normalized against proper loading controls and plotted (right bottom panel). ( B ) The INrf2Y85A mutant stabilized Nrf2. HepG2 cells were transfected wild-type INrf2–V5 or mutant INrf2Y85A–V5 for 24 hours. Cells were treated with DMSO or t -BHQ (100 μM) for the indicated periods of time. Cells were harvested and 60 μg whole-cell lysates were immunoblotted with antibodies to Nrf2, actin and NQO1 (upper panel). After immunoblotting, the intensities of NQO1 protein bands were measured and plotted (lower panel). ( C ) Luciferase assay: HepG2 cells were grown in monolayer cultures in 12-well plates for 12 hours and co-transfected with vector encoding INrf2–V5 or mutant INrf2Y85A–V5 (100 ng well −1 ) along with 100 ng well −1 of NQO1 promoter ARE–Luc reporter construct and 10 ng well −1 of firefly Renilla luciferase plasmid pRL-TK. After 24 hours of transfection, cells were treated with DMSO or t -BHQ (100 μM) for 4 to 16 hours. NQO1 promoter luciferase activity was measured and plotted as described in the Materials and Methods. The data are shown as the means ± s.d. of three independent transfection experiments.

    Journal: Journal of Cell Science

    Article Title: Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression

    doi: 10.1242/jcs.097295

    Figure Lengend Snippet: Nuclear accumulation of INrf2Y85A shows no effect on Nrf2 activation but interferes with nuclear removal of Nrf2. ( A ) Nrf2 ubiquitylation. HepG2 cells were co-transfected with constructs encoding FLAG–Nrf2, INrf2–V5 or mutant INrf2Y85A–V5 and HA–Ub for 24 hours. Cells were pretreated with MG132 (5 μM) for 2 hours and post-treated with DMSO or t -BHQ (100 μM) for the indicated periods of time. Cells were harvested and nuclear and cytosolic extracts prepared by Active Motif Kit. 500 μg of nuclear and cytosolic extracts were immunoprecipitated with antibody against FLAG and western blotted for HA–HRP antibody (left top panels). Nuclear and cytosolic ubiquitylation of FLAG–Nrf2 was measured by using Quantity-One 4.6.3 Image software (ChemiDoc XRS, Bio-Rad) and plotted (right top panels). 60 μg of nuclear lysates from the above were also immunoblotted with antibodies against FLAG, V5 and lamin-B (left bottom panel). After immunoblotting, the intensities of protein bands of nuclear FLAG–Nrf2, INrf2–V5 and mutant INrf2Y85A–V5 were measured by using Quantity-One 4.6.3. Image software, normalized against proper loading controls and plotted (right bottom panel). ( B ) The INrf2Y85A mutant stabilized Nrf2. HepG2 cells were transfected wild-type INrf2–V5 or mutant INrf2Y85A–V5 for 24 hours. Cells were treated with DMSO or t -BHQ (100 μM) for the indicated periods of time. Cells were harvested and 60 μg whole-cell lysates were immunoblotted with antibodies to Nrf2, actin and NQO1 (upper panel). After immunoblotting, the intensities of NQO1 protein bands were measured and plotted (lower panel). ( C ) Luciferase assay: HepG2 cells were grown in monolayer cultures in 12-well plates for 12 hours and co-transfected with vector encoding INrf2–V5 or mutant INrf2Y85A–V5 (100 ng well −1 ) along with 100 ng well −1 of NQO1 promoter ARE–Luc reporter construct and 10 ng well −1 of firefly Renilla luciferase plasmid pRL-TK. After 24 hours of transfection, cells were treated with DMSO or t -BHQ (100 μM) for 4 to 16 hours. NQO1 promoter luciferase activity was measured and plotted as described in the Materials and Methods. The data are shown as the means ± s.d. of three independent transfection experiments.

    Article Snippet: Antibodies used in this study were as follows: anti-INrf2 (1:1000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cul3 (1:1000), anti-Rbx1 (1:1000) purchased from Cell Signaling (Danvers, MA), anti-V5 HRP (1:5000), anti-FLAG HRP purchased from Invitrogen, anti-phosphotyrosine (1:1000) and anti-actin (1:5000) purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Activation Assay, Transfection, Construct, Mutagenesis, Immunoprecipitation, Western Blot, Software, Luciferase, Plasmid Preparation, Activity Assay

    Subcellular localization of endogenous and overexpressed INrf2. HepG2 cells were treated with chemicals for various periods of time and then harvested and nuclear and cytosolic extracts prepared. Lysates were immunoblotted. Densitometry measurements of bands were quantified and their values are shown below, relative to control DMSO measurements. Anti-LDH (cytosolic control) and anti-lamin B (nuclear control) were probed in all blots. ( A ) Cells were treated with vehicle control (DMSO) or 100 μM t -BHQ. Endogenous INrf2, Cul3 and Rbx1 were probed. ( B ) HepG2 cells were transfected with 1 μg of INrf2–V5 plasmid for 24 hours, and cells were treated with DMSO or 100 μM t -BHQ, as indicated. The lysates were immunoblotted with antibodies against V5, Cul3 and Rbx1. ( C ) Cells were pre-treated with 100 μM genistein for 2 hours, and the cells were then treated with either DMSO or 100 μM t -BHQ along with genistein for the indicated periods of time. Lysates were immunoblotted with antibodies to INrf2, Cul3 and Rbx1. ( D ) Cells were treated with 20 ng ml −1 of LMB for 2 hours; cells were then treated with either DMSO or 100 μM t -BHQ along with LMB for the indicated periods of time. Lysates were immunoblotted with antibodies to INrf2, Cul3 and Rbx1.

    Journal: Journal of Cell Science

    Article Title: Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression

    doi: 10.1242/jcs.097295

    Figure Lengend Snippet: Subcellular localization of endogenous and overexpressed INrf2. HepG2 cells were treated with chemicals for various periods of time and then harvested and nuclear and cytosolic extracts prepared. Lysates were immunoblotted. Densitometry measurements of bands were quantified and their values are shown below, relative to control DMSO measurements. Anti-LDH (cytosolic control) and anti-lamin B (nuclear control) were probed in all blots. ( A ) Cells were treated with vehicle control (DMSO) or 100 μM t -BHQ. Endogenous INrf2, Cul3 and Rbx1 were probed. ( B ) HepG2 cells were transfected with 1 μg of INrf2–V5 plasmid for 24 hours, and cells were treated with DMSO or 100 μM t -BHQ, as indicated. The lysates were immunoblotted with antibodies against V5, Cul3 and Rbx1. ( C ) Cells were pre-treated with 100 μM genistein for 2 hours, and the cells were then treated with either DMSO or 100 μM t -BHQ along with genistein for the indicated periods of time. Lysates were immunoblotted with antibodies to INrf2, Cul3 and Rbx1. ( D ) Cells were treated with 20 ng ml −1 of LMB for 2 hours; cells were then treated with either DMSO or 100 μM t -BHQ along with LMB for the indicated periods of time. Lysates were immunoblotted with antibodies to INrf2, Cul3 and Rbx1.

    Article Snippet: Antibodies used in this study were as follows: anti-INrf2 (1:1000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cul3 (1:1000), anti-Rbx1 (1:1000) purchased from Cell Signaling (Danvers, MA), anti-V5 HRP (1:5000), anti-FLAG HRP purchased from Invitrogen, anti-phosphotyrosine (1:1000) and anti-actin (1:5000) purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Transfection, Plasmid Preparation

    Immunoprecipitation of INrf2, INrf2–V5 and INrf2Y85A–V5. ( A ) HepG2 cells were treated with either DMSO or 100 μM t -BHQ for immunoprecipitations. Cells were then collected, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody against INrf2 and western blotted with antibodies to phosphotyrosine and INrf2 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibodies to INrf2 and phosphotyrosine (bottom panels). ( B , C ) HepG2 cells were transfected with 1 μg of INrf2–V5 or INrf2Y85A–V5 and treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were harvested, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to phosphotyrosine and V5 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibody to V5 (bottom panels).

    Journal: Journal of Cell Science

    Article Title: Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression

    doi: 10.1242/jcs.097295

    Figure Lengend Snippet: Immunoprecipitation of INrf2, INrf2–V5 and INrf2Y85A–V5. ( A ) HepG2 cells were treated with either DMSO or 100 μM t -BHQ for immunoprecipitations. Cells were then collected, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody against INrf2 and western blotted with antibodies to phosphotyrosine and INrf2 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibodies to INrf2 and phosphotyrosine (bottom panels). ( B , C ) HepG2 cells were transfected with 1 μg of INrf2–V5 or INrf2Y85A–V5 and treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were harvested, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to phosphotyrosine and V5 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibody to V5 (bottom panels).

    Article Snippet: Antibodies used in this study were as follows: anti-INrf2 (1:1000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cul3 (1:1000), anti-Rbx1 (1:1000) purchased from Cell Signaling (Danvers, MA), anti-V5 HRP (1:5000), anti-FLAG HRP purchased from Invitrogen, anti-phosphotyrosine (1:1000) and anti-actin (1:5000) purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Immunoprecipitation, Western Blot, Transfection

    INrf2Y85A is able to bind to Cul3–Rbx1 and Nrf2. ( A , B ) HepG2 cells were transfected with 1 μg vector encoding (A) INrf2–V5 or (B) INrf2Y85A–V5 and 500 ng vector encoding HA–Cul3 or Myc–Rbx1. Cells were then treated with 100 μM t -BHQ for the indicated periods of time. Cells were harvested; 1 mg of lysates were immunoprecipitated with antibodies against V5, HA or Myc and western blotted with antibodies against V5, HA or Myc. ( C , D ) HepG2 cells were transfected with 1 μg vector encoding (C) INrf2–V5 or (D) INrf2Y85A–V5 and pretreated with 2 μM MG132 for 16 hours. Cells were then treated with 100 μM t -BHQ for the indicated periods of time. 1 mg of lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to Nrf2 and V5 (top panels). 1 mg of lysate was immunoprecipitated with antibody to Nrf2 and western blotted with antibodies to V5 and Nrf2 (bottom panels).

    Journal: Journal of Cell Science

    Article Title: Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression

    doi: 10.1242/jcs.097295

    Figure Lengend Snippet: INrf2Y85A is able to bind to Cul3–Rbx1 and Nrf2. ( A , B ) HepG2 cells were transfected with 1 μg vector encoding (A) INrf2–V5 or (B) INrf2Y85A–V5 and 500 ng vector encoding HA–Cul3 or Myc–Rbx1. Cells were then treated with 100 μM t -BHQ for the indicated periods of time. Cells were harvested; 1 mg of lysates were immunoprecipitated with antibodies against V5, HA or Myc and western blotted with antibodies against V5, HA or Myc. ( C , D ) HepG2 cells were transfected with 1 μg vector encoding (C) INrf2–V5 or (D) INrf2Y85A–V5 and pretreated with 2 μM MG132 for 16 hours. Cells were then treated with 100 μM t -BHQ for the indicated periods of time. 1 mg of lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to Nrf2 and V5 (top panels). 1 mg of lysate was immunoprecipitated with antibody to Nrf2 and western blotted with antibodies to V5 and Nrf2 (bottom panels).

    Article Snippet: Antibodies used in this study were as follows: anti-INrf2 (1:1000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cul3 (1:1000), anti-Rbx1 (1:1000) purchased from Cell Signaling (Danvers, MA), anti-V5 HRP (1:5000), anti-FLAG HRP purchased from Invitrogen, anti-phosphotyrosine (1:1000) and anti-actin (1:5000) purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

    Cav-1 prevents MnSOD upregulation by repression of Nrf2 A. Cav-1 (green) transfected MCF7 cells demonstrate a reduction of Nrf2 (purple) and MnSOD (red). B. H 2 O 2 production in MCF7 cells expressing Cav-1 is reduced when compared to MCF7 cells devoid of Cav-1. H 2 O 2 was measured as described in methods . C. Cysteine sulfenic acid formation in MCF7 empty vector and Cav-1 expression cells using dimedone in combination with immunoprecipitation of Keap1 shows a reduction in Keap1 thiol oxidation in Cav-1 expressing MCF7 cells. D. Immunoprecipitation of Nrf2 (top) and Keap1 (bottom) demonstrates direct interactions of both proteins to Cav-1. Cells were treated with 400 μM H 2 O 2 for 1 h prior to preparation of lysates for immunoprecipitation as described in methods . E. Nrf-2 transcriptional activity was indirectly determined using NanoLuc Nrf2-stability luciferase reporter assay in MCF7 cells with and without Cav-1 stable expression in both DMEM:F12 and DMEM high glucose (4.5g/L) conditions. F. MnSOD mRNA expression following Cav-1 ectopic expression or silencing of Nrf2 in MCF7 cells shows a similar reduction in MnSOD mRNA. (Student's two-sided t -test, # = p = 0.06, * = p

    Journal: Oncotarget

    Article Title: Caveolin-1 regulates cancer cell metabolism via scavenging Nrf2 and suppressing MnSOD-driven glycolysis

    doi:

    Figure Lengend Snippet: Cav-1 prevents MnSOD upregulation by repression of Nrf2 A. Cav-1 (green) transfected MCF7 cells demonstrate a reduction of Nrf2 (purple) and MnSOD (red). B. H 2 O 2 production in MCF7 cells expressing Cav-1 is reduced when compared to MCF7 cells devoid of Cav-1. H 2 O 2 was measured as described in methods . C. Cysteine sulfenic acid formation in MCF7 empty vector and Cav-1 expression cells using dimedone in combination with immunoprecipitation of Keap1 shows a reduction in Keap1 thiol oxidation in Cav-1 expressing MCF7 cells. D. Immunoprecipitation of Nrf2 (top) and Keap1 (bottom) demonstrates direct interactions of both proteins to Cav-1. Cells were treated with 400 μM H 2 O 2 for 1 h prior to preparation of lysates for immunoprecipitation as described in methods . E. Nrf-2 transcriptional activity was indirectly determined using NanoLuc Nrf2-stability luciferase reporter assay in MCF7 cells with and without Cav-1 stable expression in both DMEM:F12 and DMEM high glucose (4.5g/L) conditions. F. MnSOD mRNA expression following Cav-1 ectopic expression or silencing of Nrf2 in MCF7 cells shows a similar reduction in MnSOD mRNA. (Student's two-sided t -test, # = p = 0.06, * = p

    Article Snippet: The membranes were blocked for 60 min at room temperature in 5% milk in TBS-T (0.05% Tween-20, pH 7.4), washed with TBS-T and then incubated with primary antibody [rabbit anti-Cav-1 (Abcam, Cambridge, MA), mouse anti-Cav-1 (BD Technologies, Research Triangle Park, NC), rabbit anti-β-actin (Cell Signaling Technologies, Boston, MA), rabbit anti-MnSOD(Abcam), rabbit anti-AMPK pThr-172 (Abcam), rabbit anti-Nrf2(Santa Cruz), mouse anti-Keap1 (Abcam), rabbit anti-Cysteine Sulfenic Acid (EMD Millipore, Billerica, MA)] in TBS-T overnight at 4°C.

    Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Activity Assay, Luciferase, Reporter Assay

    Protein expression levels of (A) Nrf2, (B) Keap1 and (C) SOD1 were examined using western blot analysis. (D) Representative western blot analysis gel using samples from in the skeletal muscles of the control, exercise, HFD and HFD + exercise groups. Western blot analysis data are presented as mean ± standard deviation. *P

    Journal: Molecular Medicine Reports

    Article Title: Chronic aerobic exercise improves insulin sensitivity and modulates Nrf2 and NF-κB/IκBα pathways in the skeletal muscle of rats fed with a high fat diet

    doi: 10.3892/mmr.2019.10787

    Figure Lengend Snippet: Protein expression levels of (A) Nrf2, (B) Keap1 and (C) SOD1 were examined using western blot analysis. (D) Representative western blot analysis gel using samples from in the skeletal muscles of the control, exercise, HFD and HFD + exercise groups. Western blot analysis data are presented as mean ± standard deviation. *P

    Article Snippet: The following proteins were assessed: NF-κB (cat# sc-372-G, Santa Cruz Biotechnology, Inc.); NF-κB inhibitor α (IκBα; cat# sc-1643, Santa Cruz Biotechnology, Inc.); interleukin (IL)-6 (cat# sc-57315, Santa Cruz Biotechnology, Inc.); nuclear factor erythroid 2-related factor 2 (Nrf2; cat# ab92946, Abcam); kelch-like ECH-associated protein 1 (Keap1; cat# ab139729, Abcam); superoxide dismutase [Cu-Zn] (SOD-1; cat# sc-271014, Santa Cruz Biotechnology, Inc.); GAPDH (cat# 2118, Cell Signaling Technology, Inc.); tripartite motif-containing family protein 72 (TRIM72; cat# ab118651, Abcam); insulin receptor substrate 1 (IRS1, cat# 3407, Cell Signaling Technology, Inc.); phosphorylated (Ser307) IRS1 (p-IRS1; cat# 2381, Cell Signaling Technology, Inc.); PI3K (cat# ab191606, Abcam); Akt (cat# 4685, Cell Signaling Technology, Inc.); phosphorylated (Ser473) Akt (p-Akt; cat# 4058, Cell Signaling Technology, Inc.); mTOR (cat# 2983, Cell Signaling Technology, Inc.); phosphorylated (Ser307) mTOR (cat# 2971, p-mTOR; Cell Signaling Technology, Inc.); and GLUT4 (cat# sc-53566, Santa Cruz Biotechnology, Inc.).

    Techniques: Expressing, Western Blot, Standard Deviation

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

    Journal: Autophagy

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

    doi: 10.4161/auto.6.5.12189

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

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

    Techniques: Knock-Out

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

    Journal: Autophagy

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

    doi: 10.4161/auto.6.5.12189

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

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

    Techniques:

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

    Journal: Autophagy

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

    doi: 10.4161/auto.6.5.12189

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

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

    Techniques: Transfection

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

    Journal: Autophagy

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

    doi: 10.4161/auto.6.5.12189

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

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

    Techniques: Knock-Out, Transfection

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

    Journal: Autophagy

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

    doi: 10.4161/auto.6.5.12189

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

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

    Techniques: Expressing, Knock-Out

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

    Journal: Autophagy

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

    doi: 10.4161/auto.6.5.12189

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

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

    Techniques: Activation Assay, Electron Microscopy, Transmission Assay

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

    Journal: Autophagy

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

    doi: 10.4161/auto.6.5.12189

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

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

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

    Antioxidant effects of SchB involved modulation of the Keap1–Nrf2 pathway. Notes: ( A ) Western blot analysis of Nrf2 following siRNA transfection in RAECs. * P

    Journal: Drug Design, Development and Therapy

    Article Title: Schisandrin B protects against angiotensin II-induced endotheliocyte deficits by targeting Keap1 and activating Nrf2 pathway

    doi: 10.2147/DDDT.S184245

    Figure Lengend Snippet: Antioxidant effects of SchB involved modulation of the Keap1–Nrf2 pathway. Notes: ( A ) Western blot analysis of Nrf2 following siRNA transfection in RAECs. * P

    Article Snippet: Antibodies against Keap1 were purchased from Novus Biologicals (Littleton, CO, USA).

    Techniques: Western Blot, Transfection

    Structural and energetic analysis of SchB to the binding site of Keap1 by MD simulation. Notes: ( A ) RMSD curves for the 200 ns MD simulation. ( B ) Dynamic cross-correlations of residue fluctuations from MD simulations. ( C ) Per residue contribution of binding energy of the Keap1–SchB complex. To get a clear view, only residues in the top ten contributed are shown. ( D ) Structural analysis of the ten most contributed residues of Keap1 to SchB. Abbreviations: MD, molecular dynamics; RMSD, root-mean SD; SchB, schisandrin B.

    Journal: Drug Design, Development and Therapy

    Article Title: Schisandrin B protects against angiotensin II-induced endotheliocyte deficits by targeting Keap1 and activating Nrf2 pathway

    doi: 10.2147/DDDT.S184245

    Figure Lengend Snippet: Structural and energetic analysis of SchB to the binding site of Keap1 by MD simulation. Notes: ( A ) RMSD curves for the 200 ns MD simulation. ( B ) Dynamic cross-correlations of residue fluctuations from MD simulations. ( C ) Per residue contribution of binding energy of the Keap1–SchB complex. To get a clear view, only residues in the top ten contributed are shown. ( D ) Structural analysis of the ten most contributed residues of Keap1 to SchB. Abbreviations: MD, molecular dynamics; RMSD, root-mean SD; SchB, schisandrin B.

    Article Snippet: Antibodies against Keap1 were purchased from Novus Biologicals (Littleton, CO, USA).

    Techniques: Binding Assay