anti keap1 antibody Santa Cruz Biotechnology Search Results


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  • 85
    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: 85/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology goat anti keap1
    <t>Keap1</t> knockdown sensitizes liver cells to PA-induced toxicity, and overexpression of Keap1 mutant (Keap1 ΔCTR) protects against lipotoxicity. ( a and b ) shKeap1#4 and shLuc Hep3B cells were treated with PA at 400 μ M or vehicle (V) for 6 h. ( a ) Caspase 3/7 catalytic activity was measured using a fluorogenic assay. ( b ) Cell death was determined by trypan blue exclusion assay. ( c ) Whole-cell lysates were prepared from shKeap1#4 and shLuc Hep3B cells treated with PA at 400 or 800 μ M or vehicle (V) for 6 h. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( d ) Whole-cell lysates were prepared from WT or hepatocyte-specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes. Immunoblot analysis were performed for mKeap1, mNrf2 and β -actin. ( e ) Isolated WT or Keap1 −/− HKO primary mouse hepatocytes were treated for 24 h with PA at 400 μ M or vehicle, and apoptotic nuclei were counted after DAPI staining. ( f ) Whole-cell lysates were prepared from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated at the indicated time points with PA 400 μ M or vehicle. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and β -actin. ( g ) Cell death was determined by trypan blue exclusion assay in Keap1 ΔCTR or control Hep3B cells treated with PA at 400 μ M or vehicle for 16 h. All data are expressed as mean±S.E.M. for three experiments; *P
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    Santa Cruz Biotechnology goat anti keap1 polyclonal antibody e20
    <t>Keap1</t> knockdown sensitizes liver cells to PA-induced toxicity, and overexpression of Keap1 mutant (Keap1 ΔCTR) protects against lipotoxicity. ( a and b ) shKeap1#4 and shLuc Hep3B cells were treated with PA at 400 μ M or vehicle (V) for 6 h. ( a ) Caspase 3/7 catalytic activity was measured using a fluorogenic assay. ( b ) Cell death was determined by trypan blue exclusion assay. ( c ) Whole-cell lysates were prepared from shKeap1#4 and shLuc Hep3B cells treated with PA at 400 or 800 μ M or vehicle (V) for 6 h. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( d ) Whole-cell lysates were prepared from WT or hepatocyte-specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes. Immunoblot analysis were performed for mKeap1, mNrf2 and β -actin. ( e ) Isolated WT or Keap1 −/− HKO primary mouse hepatocytes were treated for 24 h with PA at 400 μ M or vehicle, and apoptotic nuclei were counted after DAPI staining. ( f ) Whole-cell lysates were prepared from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated at the indicated time points with PA 400 μ M or vehicle. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and β -actin. ( g ) Cell death was determined by trypan blue exclusion assay in Keap1 ΔCTR or control Hep3B cells treated with PA at 400 μ M or vehicle for 16 h. All data are expressed as mean±S.E.M. for three experiments; *P
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    91
    Santa Cruz Biotechnology rabbit anti keap1
    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, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Santa Cruz Biotechnology keap1 sc 33569 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.
    Keap1 Sc 33569 Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti keap1 h 190
    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 H 190, supplied by Santa Cruz Biotechnology, 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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    85
    Santa Cruz Biotechnology goat 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.
    Goat Anti Keap1 Polyclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology mouse antibody against keap1
    <t>Keap1,</t> MCM3, and MCM-BP form a ternary complex. ( a ) Strep-Keap1 and FLAG-MCM3 pulldown experiments from Sf9 cells co-infected with baculoviruses expressing mouse MCM-BP together with WT or interaction deficient mutant MCM3 and Keap1 as indicated. Top panels show the Western blots of indicated proteins, bottom panel the blotted membranes that were stained with colloidal gold total protein stain. 1/300th of the starting extracts (‘input’) and 1/6th of the pulldown samples was loaded on each lane. See Supplementary Fig. S6 for full-length blots. ( b ) Strep-Keap1 - FLAG-MCM3 tandem affinity purification experiment from Sf9 cells co-infected with baculoviruses expressing all six mouse MCM2-7 subunits, Keap1, and MCM-BP. Coomassie brilliant blue stained SDS-PAGE gel on the left shows eluted material from both affinity purification steps, and unbound material from the FLAG affinity step in the middle lane. Resulting complexes were further resolved by Superose 6 size exclusion chromatography, the fractions of which are shown on right gel; co-elution of molecular weight markers is indicated at the bottom. The identity of protein bands was verified by mass spectrometry.
    Mouse Antibody Against Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology mouse anti kelch like ech associated protein 1
    <t>Keap1,</t> MCM3, and MCM-BP form a ternary complex. ( a ) Strep-Keap1 and FLAG-MCM3 pulldown experiments from Sf9 cells co-infected with baculoviruses expressing mouse MCM-BP together with WT or interaction deficient mutant MCM3 and Keap1 as indicated. Top panels show the Western blots of indicated proteins, bottom panel the blotted membranes that were stained with colloidal gold total protein stain. 1/300th of the starting extracts (‘input’) and 1/6th of the pulldown samples was loaded on each lane. See Supplementary Fig. S6 for full-length blots. ( b ) Strep-Keap1 - FLAG-MCM3 tandem affinity purification experiment from Sf9 cells co-infected with baculoviruses expressing all six mouse MCM2-7 subunits, Keap1, and MCM-BP. Coomassie brilliant blue stained SDS-PAGE gel on the left shows eluted material from both affinity purification steps, and unbound material from the FLAG affinity step in the middle lane. Resulting complexes were further resolved by Superose 6 size exclusion chromatography, the fractions of which are shown on right gel; co-elution of molecular weight markers is indicated at the bottom. The identity of protein bands was verified by mass spectrometry.
    Mouse Anti Kelch Like Ech Associated Protein 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology keap1 sc 365626 antibodies
    <t>Keap1,</t> MCM3, and MCM-BP form a ternary complex. ( a ) Strep-Keap1 and FLAG-MCM3 pulldown experiments from Sf9 cells co-infected with baculoviruses expressing mouse MCM-BP together with WT or interaction deficient mutant MCM3 and Keap1 as indicated. Top panels show the Western blots of indicated proteins, bottom panel the blotted membranes that were stained with colloidal gold total protein stain. 1/300th of the starting extracts (‘input’) and 1/6th of the pulldown samples was loaded on each lane. See Supplementary Fig. S6 for full-length blots. ( b ) Strep-Keap1 - FLAG-MCM3 tandem affinity purification experiment from Sf9 cells co-infected with baculoviruses expressing all six mouse MCM2-7 subunits, Keap1, and MCM-BP. Coomassie brilliant blue stained SDS-PAGE gel on the left shows eluted material from both affinity purification steps, and unbound material from the FLAG affinity step in the middle lane. Resulting complexes were further resolved by Superose 6 size exclusion chromatography, the fractions of which are shown on right gel; co-elution of molecular weight markers is indicated at the bottom. The identity of protein bands was verified by mass spectrometry.
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    85
    Santa Cruz Biotechnology polyclonal primary antibody against keap1
    <t>Keap1,</t> MCM3, and MCM-BP form a ternary complex. ( a ) Strep-Keap1 and FLAG-MCM3 pulldown experiments from Sf9 cells co-infected with baculoviruses expressing mouse MCM-BP together with WT or interaction deficient mutant MCM3 and Keap1 as indicated. Top panels show the Western blots of indicated proteins, bottom panel the blotted membranes that were stained with colloidal gold total protein stain. 1/300th of the starting extracts (‘input’) and 1/6th of the pulldown samples was loaded on each lane. See Supplementary Fig. S6 for full-length blots. ( b ) Strep-Keap1 - FLAG-MCM3 tandem affinity purification experiment from Sf9 cells co-infected with baculoviruses expressing all six mouse MCM2-7 subunits, Keap1, and MCM-BP. Coomassie brilliant blue stained SDS-PAGE gel on the left shows eluted material from both affinity purification steps, and unbound material from the FLAG affinity step in the middle lane. Resulting complexes were further resolved by Superose 6 size exclusion chromatography, the fractions of which are shown on right gel; co-elution of molecular weight markers is indicated at the bottom. The identity of protein bands was verified by mass spectrometry.
    Polyclonal Primary Antibody Against Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti human keap1
    PTMA binds and orchestrates with TRIM 21 to regulate Nrf2 expression through <t>p62/Keap1</t> signaling. A, Immunoprecipitation study for the interaction between endogenous TRIM 21 and PTMA . Total protein lysate from BFTC 905 cells was immunoprecipitated either with PTMA or IgG (as a control), then immunoblotted with TRIM 21. B, Western blotting for TRIM 21, PTMA , and Nrf2 in several bladder cancer cells. Numeric in red indicates the ratio of protein of interest‐to‐β‐actin. C‐F, Western blotting for TRIM 21 and Nrf2 expression while knocking down or overexpression of the PTMA gene in the indicated cells. G, Western blotting for heme oxygenase‐1 ( HMOX 1) and superoxide dismutase‐2 ( SOD 2) expression in J82 cells with ectopic expression of WT PTMA and ∆ NLS PTMA . IgG, immunoglobulin G; Keap1, Kelch‐like ECH‐associated protein 1; Nrf2, nuclear factor erythroid 2‐related factor 2; PTMA , prothymosin‐α; TRIM 21, tripartite motif‐containing protein 21
    Anti Human Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology anti keap1 e20 antibodies
    PTMA binds and orchestrates with TRIM 21 to regulate Nrf2 expression through <t>p62/Keap1</t> signaling. A, Immunoprecipitation study for the interaction between endogenous TRIM 21 and PTMA . Total protein lysate from BFTC 905 cells was immunoprecipitated either with PTMA or IgG (as a control), then immunoblotted with TRIM 21. B, Western blotting for TRIM 21, PTMA , and Nrf2 in several bladder cancer cells. Numeric in red indicates the ratio of protein of interest‐to‐β‐actin. C‐F, Western blotting for TRIM 21 and Nrf2 expression while knocking down or overexpression of the PTMA gene in the indicated cells. G, Western blotting for heme oxygenase‐1 ( HMOX 1) and superoxide dismutase‐2 ( SOD 2) expression in J82 cells with ectopic expression of WT PTMA and ∆ NLS PTMA . IgG, immunoglobulin G; Keap1, Kelch‐like ECH‐associated protein 1; Nrf2, nuclear factor erythroid 2‐related factor 2; PTMA , prothymosin‐α; TRIM 21, tripartite motif‐containing protein 21
    Anti Keap1 E20 Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology anti rbx1
    Overexpression of Nrf2 up-regulates endogenous and transfected Cul3 and <t>Rbx1</t> gene expression. A , Western analysis of Flp-in T-REx 293 (293) cells or 293/FRT/FLAG-Nrf2 (FRT/FLAG-Nrf2) cells expressing tetracycline-induced FLAG-tagged Nrf2 were incubated with 4 μg/ml tetracycline (TET) for the indicated times. Cells were harvested, lysed, and probed with anti-FLAG, anti-Cul3, and anti-Rbx1. Anti-β-actin was used as loading control. Densitometry measurements of bands were quantitated and shown in graph blots below. B and C , Cul3 and Rbx1 gene expression was analyzed by real time-PCR. 293 cells or FRT/FLAG-Nrf2 cells were seeded in a monolayer and treated with 4 μg/ml tetracycline for indicated time points. RNA was extracted, converted to cDNA. 50 ng of cDNA was analyzed using primers and probes specific for Cul3 and Rbx1 mRNA. Tetracycline-induced Nrf2 expression was also confirmed using specific primers for exogenous Nrf2. NQO1 and INrf2 were used as positive control, respectively. GusB primers and probes were used as internal control. D , 293 cells or FRT/FLAG-Nrf2 cells were co-transfected with Cul3 or Rbx1 ARE plasmids and the internal control plasmid pRL-TK. Twenty-four hours after transfection, the cells were treated with 4 μg/ml tetracycline for 8 or 16 h. pGL2 vector was used as negative control. The cells were harvested and analyzed for luciferase activity. The data shown are mean ± S.D. of three independent transfection experiments.
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    93
    Santa Cruz Biotechnology anti nrf2 antibody
    Proposed <t>Nrf2-ARE</t> signaling pathway. Nrf2 is expressed constitutively in the cell and translocates directly to the nucleus following its synthesis. Following transactivation of its genes, Nrf2 is targeted for degradation by Keap1 in the nucleus, a
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    93
    Santa Cruz Biotechnology polyclonal antibody against mouse keap1
    Proposed <t>Nrf2-ARE</t> signaling pathway. Nrf2 is expressed constitutively in the cell and translocates directly to the nucleus following its synthesis. Following transactivation of its genes, Nrf2 is targeted for degradation by Keap1 in the nucleus, a
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    Image Search Results


    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

    Nrf2 signaling is regulated by several distinct mechanisms. Nrf2 activity is augmented by exogenous (i.e., xenobiotic compounds) and/or endogenous (i.e., ROS) stressors. Free Nrf2 can bind to the ARE in the nucleus and activates hundreds of cytoprotective molecules, including detoxification enzymes like GST, antioxidant enzymes (e.g., NQO1), proteasome subunits, and molecular chaperones. Nrf2 activity is largely negatively regulated by Keap1, which binds to Nrf2 and targets it for ubiquitination and proteasomal degradation. Recently, other proteins have been shown to influence Nrf2 activity either by interacting directly with Nrf2 or with Keap1. Positive regulators, including p21 and p38MAPK, directly activate Nrf2 signaling, whereas P62/SQSTM1, PALB2, WTX, and DPP3 interact with Keap1 and thereby activate Nrf2 signaling. Other negative regulators of Nrf2, including βTrCP, SIAH2, and CRIF1, directly target it for degradation independent of Keap1. CRIF1 regulates Nrf2 signaling independent of the cell’s redox status. All of these molecules may be potential targets for cancer prevention or longevity therapeutics.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of Nrf2 signaling and longevity in naturally long-lived rodents

    doi: 10.1073/pnas.1417566112

    Figure Lengend Snippet: Nrf2 signaling is regulated by several distinct mechanisms. Nrf2 activity is augmented by exogenous (i.e., xenobiotic compounds) and/or endogenous (i.e., ROS) stressors. Free Nrf2 can bind to the ARE in the nucleus and activates hundreds of cytoprotective molecules, including detoxification enzymes like GST, antioxidant enzymes (e.g., NQO1), proteasome subunits, and molecular chaperones. Nrf2 activity is largely negatively regulated by Keap1, which binds to Nrf2 and targets it for ubiquitination and proteasomal degradation. Recently, other proteins have been shown to influence Nrf2 activity either by interacting directly with Nrf2 or with Keap1. Positive regulators, including p21 and p38MAPK, directly activate Nrf2 signaling, whereas P62/SQSTM1, PALB2, WTX, and DPP3 interact with Keap1 and thereby activate Nrf2 signaling. Other negative regulators of Nrf2, including βTrCP, SIAH2, and CRIF1, directly target it for degradation independent of Keap1. CRIF1 regulates Nrf2 signaling independent of the cell’s redox status. All of these molecules may be potential targets for cancer prevention or longevity therapeutics.

    Article Snippet: A total of 1,000 μg of protein in liver homogenates was incubated with Nrf2 (Santa Cruz) and Keap1 (Santa Cruz) after these antibodies were cross-linked to agarose beads.

    Techniques: Activity Assay

    Preternaturally long-lived naked mole-rats have higher Nrf2-signaling activity than short-lived mice. ( A ) Liver tissue of naked mole-rats have higher Nrf2–ARE-binding ( n = 4) activity than that of mice ( P = 0.0001). ( B and C ) Nrf2 total protein levels are higher (B; n = 6; P = 0.0245) and Keap1 levels lower (C; P = 0.0037) in naked mole-rats. ( D and E ) Quantitative PCR similarly shows that Nrf2 transcript levels are markedly higher ( D ; P = 0.0195) and Keap1 levels strikingly lower ( E ; P = 0.0001) in the longer-lived species. These data indicate that the higher Nrf2:ARE binding activity is due to increased Nrf2 protein availability as a result of low levels of Keap1 and concomitant decreased Nrf2 ubiquitination and proteasomal degradation. Error bars represent SEM.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of Nrf2 signaling and longevity in naturally long-lived rodents

    doi: 10.1073/pnas.1417566112

    Figure Lengend Snippet: Preternaturally long-lived naked mole-rats have higher Nrf2-signaling activity than short-lived mice. ( A ) Liver tissue of naked mole-rats have higher Nrf2–ARE-binding ( n = 4) activity than that of mice ( P = 0.0001). ( B and C ) Nrf2 total protein levels are higher (B; n = 6; P = 0.0245) and Keap1 levels lower (C; P = 0.0037) in naked mole-rats. ( D and E ) Quantitative PCR similarly shows that Nrf2 transcript levels are markedly higher ( D ; P = 0.0195) and Keap1 levels strikingly lower ( E ; P = 0.0001) in the longer-lived species. These data indicate that the higher Nrf2:ARE binding activity is due to increased Nrf2 protein availability as a result of low levels of Keap1 and concomitant decreased Nrf2 ubiquitination and proteasomal degradation. Error bars represent SEM.

    Article Snippet: A total of 1,000 μg of protein in liver homogenates was incubated with Nrf2 (Santa Cruz) and Keap1 (Santa Cruz) after these antibodies were cross-linked to agarose beads.

    Techniques: Activity Assay, Mouse Assay, Binding Assay, Real-time Polymerase Chain Reaction

    Keap1 and BTrCP levels are significantly linked to rodent MLSP and likely contribute to longevity-associated up-regulation in Nrf2-signaling activity. ( A and B ) Both Keap1 ( A ; P = 0.0013) and βTrCP ( B ; P = 0.0037) were negatively correlated with MLSP in rodents ( n = 4 for each of 10 species). Both of these proteins negatively regulate Nrf2 by binding to Nrf2 and targeting it for degradation. ( C ) Using immunoprecipitation, we confirmed that Keap1 and Nrf2 were bound to Nrf2 in liver tissues of both mice and naked mole-rats ( n = 6). Error bars represent SEM.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of Nrf2 signaling and longevity in naturally long-lived rodents

    doi: 10.1073/pnas.1417566112

    Figure Lengend Snippet: Keap1 and BTrCP levels are significantly linked to rodent MLSP and likely contribute to longevity-associated up-regulation in Nrf2-signaling activity. ( A and B ) Both Keap1 ( A ; P = 0.0013) and βTrCP ( B ; P = 0.0037) were negatively correlated with MLSP in rodents ( n = 4 for each of 10 species). Both of these proteins negatively regulate Nrf2 by binding to Nrf2 and targeting it for degradation. ( C ) Using immunoprecipitation, we confirmed that Keap1 and Nrf2 were bound to Nrf2 in liver tissues of both mice and naked mole-rats ( n = 6). Error bars represent SEM.

    Article Snippet: A total of 1,000 μg of protein in liver homogenates was incubated with Nrf2 (Santa Cruz) and Keap1 (Santa Cruz) after these antibodies were cross-linked to agarose beads.

    Techniques: Activity Assay, Binding Assay, Immunoprecipitation, Mouse Assay

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

    Journal: Diabetes

    Article Title: Restoration of Nrf2 Signaling Normalizes the Regenerative Niche

    doi: 10.2337/db15-0453

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

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

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

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

    Journal: Diabetes

    Article Title: Restoration of Nrf2 Signaling Normalizes the Regenerative Niche

    doi: 10.2337/db15-0453

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

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

    Techniques: Staining

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

    Journal: Diabetes

    Article Title: Restoration of Nrf2 Signaling Normalizes the Regenerative Niche

    doi: 10.2337/db15-0453

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

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

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

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

    Journal: Diabetes

    Article Title: Restoration of Nrf2 Signaling Normalizes the Regenerative Niche

    doi: 10.2337/db15-0453

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

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

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

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

    Journal: Diabetes

    Article Title: Restoration of Nrf2 Signaling Normalizes the Regenerative Niche

    doi: 10.2337/db15-0453

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

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

    Techniques: In Vivo Imaging, Imaging

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

    Journal: Journal of Translational Medicine

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

    doi: 10.1186/s12967-020-02467-w

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

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

    Techniques: Inhibition, Western Blot, Staining

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

    Journal: Journal of Translational Medicine

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

    doi: 10.1186/s12967-020-02467-w

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

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

    Techniques: Inhibition, Multiple Displacement Amplification

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

    Journal: Journal of Translational Medicine

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

    doi: 10.1186/s12967-020-02467-w

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

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

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

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

    Journal: Journal of Translational Medicine

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

    doi: 10.1186/s12967-020-02467-w

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

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

    Techniques: Activity Assay, Western Blot, Expressing

    The effect of Keap1 knockdown, ROS inhibitors, or addition of cytokines on intracellular growth of M. avium in MDMs. ( A ) MDMs were pretreated with siKeap1 or siNTC before infection with luciferase-expressing M. avium . Bacterial numbers were quantified over time by cfu counts. Bars represent data from two independent experiments with cfu counts analyzed in triplicate. Data shown are the mean ± SEM (* P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Keap1 regulates inflammatory signaling in Mycobacterium avium-infected human macrophages

    doi: 10.1073/pnas.1423449112

    Figure Lengend Snippet: The effect of Keap1 knockdown, ROS inhibitors, or addition of cytokines on intracellular growth of M. avium in MDMs. ( A ) MDMs were pretreated with siKeap1 or siNTC before infection with luciferase-expressing M. avium . Bacterial numbers were quantified over time by cfu counts. Bars represent data from two independent experiments with cfu counts analyzed in triplicate. Data shown are the mean ± SEM (* P

    Article Snippet: Rabbit polyclonal antibody against Keap1 (cat. no. 10503–2-AP) was obtained from ProteinTech, and Keap1 antibody (sc-15246) was obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Luciferase, Expressing

    M. avium induced cellular ROS generation and recruitment of Keap1 to its phagosome. ( A ) M. avium induction of cellular ROS production in human primary macrophages detected by confocal microscopy. After a 1-h infection, ROS was analyzed by a fluorogenic marker for ROS in live cells, 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluoresceindiacetate (carboxy-H 2 DCFDA), and TBHP was used as a positive control. NAC 5 mM was used to inhibit ROS production. ( B ) Quantification of ROS induction per cell by using Imaris Cell module from three independent experiments counting at least 100 cells for each condition. ( C ) MDMs were infected with CFP- M. avium for 4 h and fixed in 2% PFA, and localization of Keap1 was assessed by immunofluorescence staining. ( D ) Quantification of Keap1 association with M. avium phagosomes at 4 and 24 h after infection by immunofluorescence from three independent experiments, counting at least 50 infected cells per condition. ( E ) Quantification of the effect of ROS inhibition on Keap1 association with M. avium phagosomes. Macrophages were pretreated with ROS inhibitors NAC and DPI, 100 µM, for 30 min, and then cells were infected for 4 h, fixed, and stained for Keap1. ( F ) Quantification of the effect of p62 siRNA knockdown (sip62) on Keap1 association with M. avium phagosomes. Macrophages were pretreated with siNTC or sip62, 20 nM, for 72 h, and then cells were infected for 4 h, fixed, and stained for Keap1. Untr, untreated. (Scale bars: A , 20 μm; C 10 μm.) Data shown are mean ± SEM (* P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Keap1 regulates inflammatory signaling in Mycobacterium avium-infected human macrophages

    doi: 10.1073/pnas.1423449112

    Figure Lengend Snippet: M. avium induced cellular ROS generation and recruitment of Keap1 to its phagosome. ( A ) M. avium induction of cellular ROS production in human primary macrophages detected by confocal microscopy. After a 1-h infection, ROS was analyzed by a fluorogenic marker for ROS in live cells, 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluoresceindiacetate (carboxy-H 2 DCFDA), and TBHP was used as a positive control. NAC 5 mM was used to inhibit ROS production. ( B ) Quantification of ROS induction per cell by using Imaris Cell module from three independent experiments counting at least 100 cells for each condition. ( C ) MDMs were infected with CFP- M. avium for 4 h and fixed in 2% PFA, and localization of Keap1 was assessed by immunofluorescence staining. ( D ) Quantification of Keap1 association with M. avium phagosomes at 4 and 24 h after infection by immunofluorescence from three independent experiments, counting at least 50 infected cells per condition. ( E ) Quantification of the effect of ROS inhibition on Keap1 association with M. avium phagosomes. Macrophages were pretreated with ROS inhibitors NAC and DPI, 100 µM, for 30 min, and then cells were infected for 4 h, fixed, and stained for Keap1. ( F ) Quantification of the effect of p62 siRNA knockdown (sip62) on Keap1 association with M. avium phagosomes. Macrophages were pretreated with siNTC or sip62, 20 nM, for 72 h, and then cells were infected for 4 h, fixed, and stained for Keap1. Untr, untreated. (Scale bars: A , 20 μm; C 10 μm.) Data shown are mean ± SEM (* P

    Article Snippet: Rabbit polyclonal antibody against Keap1 (cat. no. 10503–2-AP) was obtained from ProteinTech, and Keap1 antibody (sc-15246) was obtained from Santa Cruz Biotechnology.

    Techniques: Confocal Microscopy, Infection, Marker, Positive Control, Immunofluorescence, Staining, Inhibition

    Keap1 knockdown showed no effect on IκB and IRF protein levels and IKKβ and TBK1 mRNA expression during M. avium infection. MDMs were transfected with siKeap1, and knockdown levels were analyzed by Western blotting compared with siNTC samples 30 min, 60 min, and 4 h after M. avium infection. ( A ) Phosphorylated and total protein levels were examined with anti-phospho (p-) or anti-total (t-) antibodies at the same time points after infection for IκB. ( B ) Protein levels for some members of the IRF family. ( C ) IKKβ and TBK1 mRNA expression was analyzed by qPCR 4 h after infection. All fold induction values have been calculated relative to uninfected controls. All experiments were repeated independently n = 4–6 times from cells obtained from different donors, and data shown are the mean ± SEM. P values obtained by Student t test were not significant.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Keap1 regulates inflammatory signaling in Mycobacterium avium-infected human macrophages

    doi: 10.1073/pnas.1423449112

    Figure Lengend Snippet: Keap1 knockdown showed no effect on IκB and IRF protein levels and IKKβ and TBK1 mRNA expression during M. avium infection. MDMs were transfected with siKeap1, and knockdown levels were analyzed by Western blotting compared with siNTC samples 30 min, 60 min, and 4 h after M. avium infection. ( A ) Phosphorylated and total protein levels were examined with anti-phospho (p-) or anti-total (t-) antibodies at the same time points after infection for IκB. ( B ) Protein levels for some members of the IRF family. ( C ) IKKβ and TBK1 mRNA expression was analyzed by qPCR 4 h after infection. All fold induction values have been calculated relative to uninfected controls. All experiments were repeated independently n = 4–6 times from cells obtained from different donors, and data shown are the mean ± SEM. P values obtained by Student t test were not significant.

    Article Snippet: Rabbit polyclonal antibody against Keap1 (cat. no. 10503–2-AP) was obtained from ProteinTech, and Keap1 antibody (sc-15246) was obtained from Santa Cruz Biotechnology.

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

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

    Journal: Cell Death and Differentiation

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

    doi: 10.1038/cdd.2014.49

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

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

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

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

    Journal: Cell Death and Differentiation

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

    doi: 10.1038/cdd.2014.49

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

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

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

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

    Journal: Cell Death and Differentiation

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

    doi: 10.1038/cdd.2014.49

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

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

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

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

    Journal: Cell Death and Differentiation

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

    doi: 10.1038/cdd.2014.49

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

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

    Techniques: Stable Transfection, Expressing, Trypan Blue Exclusion Assay

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

    Journal: Cell Death and Differentiation

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

    doi: 10.1038/cdd.2014.49

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

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

    Techniques: Stable Transfection, Expressing, Trypan Blue Exclusion Assay

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

    Journal: Cell Death and Differentiation

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

    doi: 10.1038/cdd.2014.49

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

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

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

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

    Journal: Cell Death and Differentiation

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

    doi: 10.1038/cdd.2014.49

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

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

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

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

    Journal: Cell Death and Differentiation

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

    doi: 10.1038/cdd.2014.49

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

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

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

    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: For immunofluorescence sections were probed with rabbit anti-Keap1 (Santa Cruz, #33569; 1:500 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: For immunofluorescence sections were probed with rabbit anti-Keap1 (Santa Cruz, #33569; 1:500 dilution).

    Techniques: Expressing, Immunofluorescence, Staining

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.25.11.4501-4513.2005

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

    Article Snippet: For immunostaining, rabbit anti-Keap1 antibody ( ) was used at 10 μg/ml, rabbit anti-Nrf2 antibody (Santa Cruz, Paso Robles, CA) was used at 1:100, mouse anti-vinculin antibody (Sigma-Aldrich, St. Louis, MO) was used at 1:400, mouse anti-β-catenin antibody (BD Biosciences, San Diego, CA) was used at 1:50, rabbit anti-glutathione S -transferase (GST) alpha (Alpha Diagnostic, Inc., San Antonio, TX) was used at 1:50.

    Techniques: Inhibition, Translocation Assay, Activation Assay

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.25.11.4501-4513.2005

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

    Article Snippet: For immunostaining, rabbit anti-Keap1 antibody ( ) was used at 10 μg/ml, rabbit anti-Nrf2 antibody (Santa Cruz, Paso Robles, CA) was used at 1:100, mouse anti-vinculin antibody (Sigma-Aldrich, St. Louis, MO) was used at 1:400, mouse anti-β-catenin antibody (BD Biosciences, San Diego, CA) was used at 1:50, rabbit anti-glutathione S -transferase (GST) alpha (Alpha Diagnostic, Inc., San Antonio, TX) was used at 1:50.

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

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.25.11.4501-4513.2005

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

    Article Snippet: For immunostaining, rabbit anti-Keap1 antibody ( ) was used at 10 μg/ml, rabbit anti-Nrf2 antibody (Santa Cruz, Paso Robles, CA) was used at 1:100, mouse anti-vinculin antibody (Sigma-Aldrich, St. Louis, MO) was used at 1:400, mouse anti-β-catenin antibody (BD Biosciences, San Diego, CA) was used at 1:50, rabbit anti-glutathione S -transferase (GST) alpha (Alpha Diagnostic, Inc., San Antonio, TX) was used at 1:50.

    Techniques: Construct, Fluorescence, Staining

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-30562-y

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

    Article Snippet: Goat antibody against MCM3 (N19, sc-9850) and mouse antibody against Keap1 (sc-365626; both from Santa Cruz Biotechnology, Inc.) were used as primary probes at 1:50 dilution and incubated at 4 °C overnight.

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-30562-y

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

    Article Snippet: Goat antibody against MCM3 (N19, sc-9850) and mouse antibody against Keap1 (sc-365626; both from Santa Cruz Biotechnology, Inc.) were used as primary probes at 1:50 dilution and incubated at 4 °C overnight.

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-30562-y

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

    Article Snippet: Goat antibody against MCM3 (N19, sc-9850) and mouse antibody against Keap1 (sc-365626; both from Santa Cruz Biotechnology, Inc.) were used as primary probes at 1:50 dilution and incubated at 4 °C overnight.

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-30562-y

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

    Article Snippet: Goat antibody against MCM3 (N19, sc-9850) and mouse antibody against Keap1 (sc-365626; both from Santa Cruz Biotechnology, Inc.) were used as primary probes at 1:50 dilution and incubated at 4 °C overnight.

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-30562-y

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

    Article Snippet: Goat antibody against MCM3 (N19, sc-9850) and mouse antibody against Keap1 (sc-365626; both from Santa Cruz Biotechnology, Inc.) were used as primary probes at 1:50 dilution and incubated at 4 °C overnight.

    Techniques: Sequencing

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-30562-y

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

    Article Snippet: Goat antibody against MCM3 (N19, sc-9850) and mouse antibody against Keap1 (sc-365626; both from Santa Cruz Biotechnology, Inc.) were used as primary probes at 1:50 dilution and incubated at 4 °C overnight.

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-30562-y

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

    Article Snippet: Goat antibody against MCM3 (N19, sc-9850) and mouse antibody against Keap1 (sc-365626; both from Santa Cruz Biotechnology, Inc.) were used as primary probes at 1:50 dilution and incubated at 4 °C overnight.

    Techniques: Sequencing

    Siah2, but not Keap1, knock-down inhibits hypoglycemia-induced Nrf2 down-regulation and restores endothelial monolayer integrity. (A) Gene silencing efficiency and specificity determined by IF staining and western blot analysis of Siah2 (red) and Keap1 (green) in hCMEC/D3 cells transfected with gene specific or scramble siRNA. Respective bands with β-actin as loading control were shown at the bottom of the graph (n = 3/condition). (B) IF and western blot analyses of Nrf2 (red) expression/distribution in scramble and Siah2 or Keap1 transfected hCMEC/D3 cells exposed to normal or hypoglycemic conditions (12h) after 72h following transfection (n = 3/condition). (C) Paracellular permeability to labeled dextrans of variable size (4-70kDa) across hCMEC/D3 monolayers transfected with scramble or Siah2-specific siRNA and exposed to 12h normal or hypoglycemia following an interval of 72h after transfection (n = 4-6/condition). ( D ) Hypoglycemia-induced increase in dextran permeability is independent of the osmotic effects of the media. HCMEC/D3 cells were exposed to equimolar concentrations of glucose with normalglycemic media (L-normal) containing 5.5mM D-glucose + 4.5mM L-glucose and hypoglycemic media (L-Hypo) containing 2.2mM D-glucose + 7.8mM L-glucose (n = 4-5/condition). Images were captured at 40X (scale: 100 μm) and merged with DAPI. Data were expressed as mean ± SEM (% of scramble control). *** P

    Journal: PLoS ONE

    Article Title: Altered Nrf2 Signaling Mediates Hypoglycemia-Induced Blood–Brain Barrier Endothelial Dysfunction In Vitro

    doi: 10.1371/journal.pone.0122358

    Figure Lengend Snippet: Siah2, but not Keap1, knock-down inhibits hypoglycemia-induced Nrf2 down-regulation and restores endothelial monolayer integrity. (A) Gene silencing efficiency and specificity determined by IF staining and western blot analysis of Siah2 (red) and Keap1 (green) in hCMEC/D3 cells transfected with gene specific or scramble siRNA. Respective bands with β-actin as loading control were shown at the bottom of the graph (n = 3/condition). (B) IF and western blot analyses of Nrf2 (red) expression/distribution in scramble and Siah2 or Keap1 transfected hCMEC/D3 cells exposed to normal or hypoglycemic conditions (12h) after 72h following transfection (n = 3/condition). (C) Paracellular permeability to labeled dextrans of variable size (4-70kDa) across hCMEC/D3 monolayers transfected with scramble or Siah2-specific siRNA and exposed to 12h normal or hypoglycemia following an interval of 72h after transfection (n = 4-6/condition). ( D ) Hypoglycemia-induced increase in dextran permeability is independent of the osmotic effects of the media. HCMEC/D3 cells were exposed to equimolar concentrations of glucose with normalglycemic media (L-normal) containing 5.5mM D-glucose + 4.5mM L-glucose and hypoglycemic media (L-Hypo) containing 2.2mM D-glucose + 7.8mM L-glucose (n = 4-5/condition). Images were captured at 40X (scale: 100 μm) and merged with DAPI. Data were expressed as mean ± SEM (% of scramble control). *** P

    Article Snippet: Antibodies were obtained from the following sources: Rabbit anti-ZO-1 (#D7D12), anti-VE-cadherin (#D87F2), and goat anti-mouse (#4408S) and anti-rabbit (#4413S) conjugated to Alexa Fluor 488 and 555 from Cell Signaling Technology (Danvers, MA, USA); mouse anti-β actin (#A5441) and anti-Siah2 (S7945) from Sigma-Aldrich; rabbit anti-Nrf2 (#sc-722), mouse anti-NQO1 (#sc-271116), mouse anti-Keap1 (#sc-365626) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); donkey anti-rabbit (#NA934) and sheep anti-mouse (#NA931) HRP-linked antibodies from GE Healthcare (Piscataway, NJ, USA).

    Techniques: Staining, Western Blot, Transfection, Expressing, Permeability, Labeling

    Hypoglycemia induces progressive down-regulation of Nrf2 expression (protein) and function in hCMEC/D3 cells. (A) IF and western blot analyses of BBB endothelial Nrf2 and its downstream target, NQO1, expression and distribution following 3-24h exposure to normal or hypoglycemic media (see Methods ; n = 3-4/condition). Respective bands with β-actin as loading control were shown above the graphs for each time point. (B) Effects of hypoglycemia on protein expression/distribution of intracellular regulators of Nrf2, such as Siah2 (3-12h) and Keap1 (12h; B2 ), as assessed by IF and western blots (n = 3-4/condition). Further, a magnified view of the region represented by yellow box was provided in the inset to demonstrate the cellular localization changes of Siah2 following 12 h exposure to control or hypoglycemic conditions. (C) Real-time qRT-PCR based analysis of mRNA expression of target genes in hCMEC/D3 cells exposed to normal or hypoglycemic media (12h) (n = 4/condition). Data were expressed as mean ± SEM (% normalglycemic control for western blots) or fold change over control (mRNA expression). Images were captured at 40X (scale: 100μm) and merged with DAPI. * P

    Journal: PLoS ONE

    Article Title: Altered Nrf2 Signaling Mediates Hypoglycemia-Induced Blood–Brain Barrier Endothelial Dysfunction In Vitro

    doi: 10.1371/journal.pone.0122358

    Figure Lengend Snippet: Hypoglycemia induces progressive down-regulation of Nrf2 expression (protein) and function in hCMEC/D3 cells. (A) IF and western blot analyses of BBB endothelial Nrf2 and its downstream target, NQO1, expression and distribution following 3-24h exposure to normal or hypoglycemic media (see Methods ; n = 3-4/condition). Respective bands with β-actin as loading control were shown above the graphs for each time point. (B) Effects of hypoglycemia on protein expression/distribution of intracellular regulators of Nrf2, such as Siah2 (3-12h) and Keap1 (12h; B2 ), as assessed by IF and western blots (n = 3-4/condition). Further, a magnified view of the region represented by yellow box was provided in the inset to demonstrate the cellular localization changes of Siah2 following 12 h exposure to control or hypoglycemic conditions. (C) Real-time qRT-PCR based analysis of mRNA expression of target genes in hCMEC/D3 cells exposed to normal or hypoglycemic media (12h) (n = 4/condition). Data were expressed as mean ± SEM (% normalglycemic control for western blots) or fold change over control (mRNA expression). Images were captured at 40X (scale: 100μm) and merged with DAPI. * P

    Article Snippet: Antibodies were obtained from the following sources: Rabbit anti-ZO-1 (#D7D12), anti-VE-cadherin (#D87F2), and goat anti-mouse (#4408S) and anti-rabbit (#4413S) conjugated to Alexa Fluor 488 and 555 from Cell Signaling Technology (Danvers, MA, USA); mouse anti-β actin (#A5441) and anti-Siah2 (S7945) from Sigma-Aldrich; rabbit anti-Nrf2 (#sc-722), mouse anti-NQO1 (#sc-271116), mouse anti-Keap1 (#sc-365626) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); donkey anti-rabbit (#NA934) and sheep anti-mouse (#NA931) HRP-linked antibodies from GE Healthcare (Piscataway, NJ, USA).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR

    PTMA binds and orchestrates with TRIM 21 to regulate Nrf2 expression through p62/Keap1 signaling. A, Immunoprecipitation study for the interaction between endogenous TRIM 21 and PTMA . Total protein lysate from BFTC 905 cells was immunoprecipitated either with PTMA or IgG (as a control), then immunoblotted with TRIM 21. B, Western blotting for TRIM 21, PTMA , and Nrf2 in several bladder cancer cells. Numeric in red indicates the ratio of protein of interest‐to‐β‐actin. C‐F, Western blotting for TRIM 21 and Nrf2 expression while knocking down or overexpression of the PTMA gene in the indicated cells. G, Western blotting for heme oxygenase‐1 ( HMOX 1) and superoxide dismutase‐2 ( SOD 2) expression in J82 cells with ectopic expression of WT PTMA and ∆ NLS PTMA . IgG, immunoglobulin G; Keap1, Kelch‐like ECH‐associated protein 1; Nrf2, nuclear factor erythroid 2‐related factor 2; PTMA , prothymosin‐α; TRIM 21, tripartite motif‐containing protein 21

    Journal: Cancer Science

    Article Title: Prothymosin‐α enhances phosphatase and tensin homolog expression and binds with tripartite motif‐containing protein 21 to regulate Kelch‐like ECH‐associated protein 1/nuclear factor erythroid 2‐related factor 2 signaling in human bladder cancer. Prothymosin‐α enhances phosphatase and tensin homolog expression and binds with tripartite motif‐containing protein 21 to regulate Kelch‐like ECH‐associated protein 1/nuclear factor erythroid 2‐related factor 2 signaling in human bladder cancer

    doi: 10.1111/cas.13963

    Figure Lengend Snippet: PTMA binds and orchestrates with TRIM 21 to regulate Nrf2 expression through p62/Keap1 signaling. A, Immunoprecipitation study for the interaction between endogenous TRIM 21 and PTMA . Total protein lysate from BFTC 905 cells was immunoprecipitated either with PTMA or IgG (as a control), then immunoblotted with TRIM 21. B, Western blotting for TRIM 21, PTMA , and Nrf2 in several bladder cancer cells. Numeric in red indicates the ratio of protein of interest‐to‐β‐actin. C‐F, Western blotting for TRIM 21 and Nrf2 expression while knocking down or overexpression of the PTMA gene in the indicated cells. G, Western blotting for heme oxygenase‐1 ( HMOX 1) and superoxide dismutase‐2 ( SOD 2) expression in J82 cells with ectopic expression of WT PTMA and ∆ NLS PTMA . IgG, immunoglobulin G; Keap1, Kelch‐like ECH‐associated protein 1; Nrf2, nuclear factor erythroid 2‐related factor 2; PTMA , prothymosin‐α; TRIM 21, tripartite motif‐containing protein 21

    Article Snippet: Protein (30 μg) from each sample was subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), transferred onto nitrocellulose membrane filter, and subsequently immunoblotted with anti‐human PTMA (4F4, ALX‐804‐487; 2F11, ALX‐804‐486; Alexis, Lausen, Switzerland), anti‐human TRIM21 (GTX113554, Genetex, Irvine, CA, USA), anti‐human PTEN (6H2.1, #04‐035; Millipore, Burlington, MA, USA), anti‐human Nrf2 (C‐20, sc‐722; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐human Keap1 (E‐20, sc‐15246, Santa Cruz), anti‐human p62 (D‐3, sc‐28359, Santa Cruz), anti‐Ubiquitin (FL‐76, sc‐9133; Santa Cruz Biotechnology), anti‐superoxide dismutase‐2 (GTX116093; GeneTex) and anti‐heme oxygenase‐1 (GTX101147; Genetex). β‐Actin protein served as an internal control.

    Techniques: Expressing, Immunoprecipitation, Western Blot, Over Expression

    Overexpression of Nrf2 up-regulates endogenous and transfected Cul3 and Rbx1 gene expression. A , Western analysis of Flp-in T-REx 293 (293) cells or 293/FRT/FLAG-Nrf2 (FRT/FLAG-Nrf2) cells expressing tetracycline-induced FLAG-tagged Nrf2 were incubated with 4 μg/ml tetracycline (TET) for the indicated times. Cells were harvested, lysed, and probed with anti-FLAG, anti-Cul3, and anti-Rbx1. Anti-β-actin was used as loading control. Densitometry measurements of bands were quantitated and shown in graph blots below. B and C , Cul3 and Rbx1 gene expression was analyzed by real time-PCR. 293 cells or FRT/FLAG-Nrf2 cells were seeded in a monolayer and treated with 4 μg/ml tetracycline for indicated time points. RNA was extracted, converted to cDNA. 50 ng of cDNA was analyzed using primers and probes specific for Cul3 and Rbx1 mRNA. Tetracycline-induced Nrf2 expression was also confirmed using specific primers for exogenous Nrf2. NQO1 and INrf2 were used as positive control, respectively. GusB primers and probes were used as internal control. D , 293 cells or FRT/FLAG-Nrf2 cells were co-transfected with Cul3 or Rbx1 ARE plasmids and the internal control plasmid pRL-TK. Twenty-four hours after transfection, the cells were treated with 4 μg/ml tetracycline for 8 or 16 h. pGL2 vector was used as negative control. The cells were harvested and analyzed for luciferase activity. The data shown are mean ± S.D. of three independent transfection experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: An Autoregulatory Loop between Nrf2 and Cul3-Rbx1 Controls Their Cellular Abundance *

    doi: 10.1074/jbc.M110.121863

    Figure Lengend Snippet: Overexpression of Nrf2 up-regulates endogenous and transfected Cul3 and Rbx1 gene expression. A , Western analysis of Flp-in T-REx 293 (293) cells or 293/FRT/FLAG-Nrf2 (FRT/FLAG-Nrf2) cells expressing tetracycline-induced FLAG-tagged Nrf2 were incubated with 4 μg/ml tetracycline (TET) for the indicated times. Cells were harvested, lysed, and probed with anti-FLAG, anti-Cul3, and anti-Rbx1. Anti-β-actin was used as loading control. Densitometry measurements of bands were quantitated and shown in graph blots below. B and C , Cul3 and Rbx1 gene expression was analyzed by real time-PCR. 293 cells or FRT/FLAG-Nrf2 cells were seeded in a monolayer and treated with 4 μg/ml tetracycline for indicated time points. RNA was extracted, converted to cDNA. 50 ng of cDNA was analyzed using primers and probes specific for Cul3 and Rbx1 mRNA. Tetracycline-induced Nrf2 expression was also confirmed using specific primers for exogenous Nrf2. NQO1 and INrf2 were used as positive control, respectively. GusB primers and probes were used as internal control. D , 293 cells or FRT/FLAG-Nrf2 cells were co-transfected with Cul3 or Rbx1 ARE plasmids and the internal control plasmid pRL-TK. Twenty-four hours after transfection, the cells were treated with 4 μg/ml tetracycline for 8 or 16 h. pGL2 vector was used as negative control. The cells were harvested and analyzed for luciferase activity. The data shown are mean ± S.D. of three independent transfection experiments.

    Article Snippet: The membranes were incubated with anti-Cul3 (1:1000, Cell Signaling), anti-Rbx1 (1:1000, Bio Source) anti-INrf2 (1:1000, Santa Cruz Biotechnology), anti-Nrf2 (1:500, Santa Cruz Biotechnology), anti-FLAG (1:5000, Sigma), anti-V5 (1:5000, Invitrogen), anti-Myc (1:5000, Sigma), or anti-actin (1:5000, Sigma) antibodies, washed, and probed with electrochemiluminescence (Amersham Biosciences).

    Techniques: Over Expression, Transfection, Expressing, Western Blot, Incubation, Real-time Polymerase Chain Reaction, Positive Control, Plasmid Preparation, Negative Control, Luciferase, Activity Assay

    Autofeedback loop between Nrf2 and Cul3-Rbx1. A model that demonstrates an autoregulatory loop between Cul3-Rbx1 and Nrf2 is shown. The Nrf2 protein regulates the Cul3-Rbx1 genes at the level of transcription, and the Cul3-Rbx1 protein regulates the Nrf2 protein at the level of its activity.

    Journal: The Journal of Biological Chemistry

    Article Title: An Autoregulatory Loop between Nrf2 and Cul3-Rbx1 Controls Their Cellular Abundance *

    doi: 10.1074/jbc.M110.121863

    Figure Lengend Snippet: Autofeedback loop between Nrf2 and Cul3-Rbx1. A model that demonstrates an autoregulatory loop between Cul3-Rbx1 and Nrf2 is shown. The Nrf2 protein regulates the Cul3-Rbx1 genes at the level of transcription, and the Cul3-Rbx1 protein regulates the Nrf2 protein at the level of its activity.

    Article Snippet: The membranes were incubated with anti-Cul3 (1:1000, Cell Signaling), anti-Rbx1 (1:1000, Bio Source) anti-INrf2 (1:1000, Santa Cruz Biotechnology), anti-Nrf2 (1:500, Santa Cruz Biotechnology), anti-FLAG (1:5000, Sigma), anti-V5 (1:5000, Invitrogen), anti-Myc (1:5000, Sigma), or anti-actin (1:5000, Sigma) antibodies, washed, and probed with electrochemiluminescence (Amersham Biosciences).

    Techniques: Activity Assay

    Proposed Nrf2-ARE signaling pathway. Nrf2 is expressed constitutively in the cell and translocates directly to the nucleus following its synthesis. Following transactivation of its genes, Nrf2 is targeted for degradation by Keap1 in the nucleus, a

    Journal: The Journal of Biological Chemistry

    Article Title: The Nrf2-Antioxidant Response Element Signaling Pathway and Its Activation by Oxidative Stress *

    doi: 10.1074/jbc.R900010200

    Figure Lengend Snippet: Proposed Nrf2-ARE signaling pathway. Nrf2 is expressed constitutively in the cell and translocates directly to the nucleus following its synthesis. Following transactivation of its genes, Nrf2 is targeted for degradation by Keap1 in the nucleus, a

    Article Snippet: Nrf2 was stained with an anti-Nrf2 antibody ( An important question is how Keap1 targets Nrf2 for ubiquitylation, given their localization in distinct cellular compartments.

    Techniques:

    Regulation of rat GSTA2 and NQO1 gene expression. Induction of these two detoxication enzymes is regulated at the transcriptional level mediated by two distinct enhancers, XRE and ARE, controlled by the AhR and Nrf2, respectively. β-Naphthoflavone

    Journal: The Journal of Biological Chemistry

    Article Title: The Nrf2-Antioxidant Response Element Signaling Pathway and Its Activation by Oxidative Stress *

    doi: 10.1074/jbc.R900010200

    Figure Lengend Snippet: Regulation of rat GSTA2 and NQO1 gene expression. Induction of these two detoxication enzymes is regulated at the transcriptional level mediated by two distinct enhancers, XRE and ARE, controlled by the AhR and Nrf2, respectively. β-Naphthoflavone

    Article Snippet: Nrf2 was stained with an anti-Nrf2 antibody ( An important question is how Keap1 targets Nrf2 for ubiquitylation, given their localization in distinct cellular compartments.

    Techniques: Expressing

    Nuclear localization of Nrf2 in human umbilical vein endothelial cells. ) and visualized with a secondary antibody conjugated

    Journal: The Journal of Biological Chemistry

    Article Title: The Nrf2-Antioxidant Response Element Signaling Pathway and Its Activation by Oxidative Stress *

    doi: 10.1074/jbc.R900010200

    Figure Lengend Snippet: Nuclear localization of Nrf2 in human umbilical vein endothelial cells. ) and visualized with a secondary antibody conjugated

    Article Snippet: Nrf2 was stained with an anti-Nrf2 antibody ( An important question is how Keap1 targets Nrf2 for ubiquitylation, given their localization in distinct cellular compartments.

    Techniques: