anti keap1 antibody Santa Cruz Biotechnology Search Results


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  • 79
    Santa Cruz Biotechnology anti inrf2
    Cul3–Rbx1 export out of the nucleus by means of the <t>INrf2</t> nuclear export signal. ( A , B ) Immunocytochemistry: HepG2 cells (A) or Hepa-1 cells (B) were grown in Lab-Tek II chamber slides. Cells were fixed, permeabilized and incubated with a 1:500 dilution of anti-goat INrf2, anti-rabbit Cul3 and anti-rabbit Rbx1 antibody, as indicated in the figures. Cells were washed and incubated with Alexa-Fluor-594-conjugated anti-goat antibody or FITC-conjugated anti-rabbit secondary antibody (Invitrogen). Cells were washed twice with PBS, stained with Vectashield containing nuclear DAPI stain, observed under a Nikon fluorescence microscope and photographed. ( C ) Hepa-1 cells were transfected with INrf2 siRNA for 48 hours. Cells were then treated with 100 μM t -BHQ for the indicated periods of time and harvested. Cytosolic and nuclear extracts were prepared, and lysates were immunoblotted with antibodies against INrf2, Cul3, Rbx1, LDH and lamin B. ( D ) Schematic diagram of the mouse INrf2 gene showing the BTB domain (responsible for Cul3 interaction), DGR domain (responsible for Nrf2 interaction) and a functional nuclear export signal (NES). ( E ) HepG2 cells were treated and transfected with 1 μg of vector encoding INrf2ΔNES–V5. Cells were then treated with 100 μM t -BHQ, harvested and nuclear and cytosolic extracts were then prepared. Lysates were immunoblotted with antibodies against V5, Cul3, Rbx1, lamin B and LDH. ( F ) HepG2 cells were co-transfected with 1 μg of INrf2–V5 or INrf2Y85A–V5 and 1 μg of FLAG–Crm1 and then treated with either DMSO or 100 μM t -BHQ for the indicated periods of time (left and right panels). Cells were harvested; 1 mg of lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to FLAG and V5 (top two panels). 1 mg of lysate was immunoprecipitated with antibody to FLAG and western blotted with antibodies to V5 and FLAG (bottom two panels).
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    Santa Cruz Biotechnology keap1 sc 33569
    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).
    Keap1 Sc 33569, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology goat anti keap1
    Acute proteasome stress co-opts SQSTM1 onto protein aggregates, and induces de novo SQSTM1 expression. ( A, B ) Immunofluorescence analysis of SQSTM1 and ubiquitin accumulation in MM lines upon treatment with bortezomib (Btz). Nuclei are stained blue with DAPI. Scale bars: 10 µm. ( A ) SQSTM1 in MM.1S cells left untreated (left) or treated for 1 h with 1 µM Btz (right) (n = 5 independent experiments). ( B ) SQSTM1 and ubiquitin in MM.1S cells treated with Btz (as in A ). ( C ) Co-immunoprecipitation (IP) of polyubiquitinated proteins with SQSTM1. MM.1S cells were treated with Btz (as in A ), prior to IP of SQSTM1, and the association of ubiquitinated proteins and <t>KEAP1</t> with SQSTM1 assessed by immunoblot (n ≥ 3 ). ( D ) Changes of selected proteins of the SQSTM1 interactome upon treatment with Btz (as in A ) as determined by SILAC LC-MS/MS in MM.1S cells (more proteins listed in Table 1 ). ( E ) Quantitative RT-PCR analysis of transcripts encoding the indicated autophagy receptors in MM lines treated with 1 µM Btz for 4 h. mRNA amounts were normalized by histone H3 and expressed relative to untreated controls (average induction ±s.e.m.; n = 3). ( F ) Immunoblot analysis of SQSTM1 and LC3 in the indicated MM lines treated with 1 µM Btz for 8 h (representative blot, n = 3). ( G ) Immunoblot analysis of SQSTM1 in MM.1S cells treated with 1 µM Btz for 8 h in the presence or absence of 10 µg/ml cycloheximide (CHX).
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    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.
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    Millipore keap1
    p62/SQSTM1 phosphorylation at Ser351 and <t>Keap1</t> downregulation in sodium arsenite-treated human normal and HGPS fibroblasts A) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. Intracellular ROS were detected after incubation with CM-H2DCFDA and flow cytometry. The results are mean + sd. of three independent experiments. The ROS level in untreated normal fibroblasts was defined as 1.0. B) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. 5 μg of cytoplasmic extracts were loaded on 10% SDS-PAGE and expression of Keap1, p62/SQSTM1, phosphorylated p62/SQSTM1 at Ser351 were analyzed by Western blotting. β-actin is shown as a loading control of each sample.
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    Santa Cruz Biotechnology keap1 sc 365626 antibodies
    p62/SQSTM1 phosphorylation at Ser351 and <t>Keap1</t> downregulation in sodium arsenite-treated human normal and HGPS fibroblasts A) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. Intracellular ROS were detected after incubation with CM-H2DCFDA and flow cytometry. The results are mean + sd. of three independent experiments. The ROS level in untreated normal fibroblasts was defined as 1.0. B) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. 5 μg of cytoplasmic extracts were loaded on 10% SDS-PAGE and expression of Keap1, p62/SQSTM1, phosphorylated p62/SQSTM1 at Ser351 were analyzed by Western blotting. β-actin is shown as a loading control of each sample.
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    Santa Cruz Biotechnology anti keap1 polyclonal antibody
    p62/SQSTM1 phosphorylation at Ser351 and <t>Keap1</t> downregulation in sodium arsenite-treated human normal and HGPS fibroblasts A) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. Intracellular ROS were detected after incubation with CM-H2DCFDA and flow cytometry. The results are mean + sd. of three independent experiments. The ROS level in untreated normal fibroblasts was defined as 1.0. B) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. 5 μg of cytoplasmic extracts were loaded on 10% SDS-PAGE and expression of Keap1, p62/SQSTM1, phosphorylated p62/SQSTM1 at Ser351 were analyzed by Western blotting. β-actin is shown as a loading control of each sample.
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    Santa Cruz Biotechnology anti keap1 e20 antibodies
    p62/SQSTM1 phosphorylation at Ser351 and <t>Keap1</t> downregulation in sodium arsenite-treated human normal and HGPS fibroblasts A) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. Intracellular ROS were detected after incubation with CM-H2DCFDA and flow cytometry. The results are mean + sd. of three independent experiments. The ROS level in untreated normal fibroblasts was defined as 1.0. B) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. 5 μg of cytoplasmic extracts were loaded on 10% SDS-PAGE and expression of Keap1, p62/SQSTM1, phosphorylated p62/SQSTM1 at Ser351 were analyzed by Western blotting. β-actin is shown as a loading control of each sample.
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    Santa Cruz Biotechnology polyclonal keap1 e20 antibody
    p62/SQSTM1 phosphorylation at Ser351 and <t>Keap1</t> downregulation in sodium arsenite-treated human normal and HGPS fibroblasts A) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. Intracellular ROS were detected after incubation with CM-H2DCFDA and flow cytometry. The results are mean + sd. of three independent experiments. The ROS level in untreated normal fibroblasts was defined as 1.0. B) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. 5 μg of cytoplasmic extracts were loaded on 10% SDS-PAGE and expression of Keap1, p62/SQSTM1, phosphorylated p62/SQSTM1 at Ser351 were analyzed by Western blotting. β-actin is shown as a loading control of each sample.
    Polyclonal Keap1 E20 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology goat polyclonal anti keap1 antibody
    p62/SQSTM1 phosphorylation at Ser351 and <t>Keap1</t> downregulation in sodium arsenite-treated human normal and HGPS fibroblasts A) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. Intracellular ROS were detected after incubation with CM-H2DCFDA and flow cytometry. The results are mean + sd. of three independent experiments. The ROS level in untreated normal fibroblasts was defined as 1.0. B) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. 5 μg of cytoplasmic extracts were loaded on 10% SDS-PAGE and expression of Keap1, p62/SQSTM1, phosphorylated p62/SQSTM1 at Ser351 were analyzed by Western blotting. β-actin is shown as a loading control of each sample.
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    Santa Cruz Biotechnology mouse monoclonal anti keap1
    SQSTM1 accumulation activates the <t>SQSTM1-KEAP1-NFE2L2</t> axis that reduces ROS production in EBV-infected monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) KEAP1 and NFE2L2 expression by western blot, (b) NFE2L2 localization by IFA and (c) CAT and GSR expression by western blot. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of KEAP1:ACTB, NFE2L2:ACTB, CAT:ACTB, GSR:ACTB of 3 different experiments. SQSTM1 staining is shown in red; bars: 10 mm. Differentiating monocytes exposed to EBV were silenced for si SQSTM1 with specific siRNA or scrambled siRNA and cultured for 5 days with CSF2 and IL4 before analysing (d) NFE2L2 localization by IFA and (e) SQSTM1, CAT and GSR expression by western blot. NFE2L2 staining is shown in red; bars: 10 mm. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of SQSTM1:ACTB, CAT:ACTB, GSR:ACTB of 3 different experiments. * P value
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    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: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit anti keap1 polyclonal antibody
    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
    Rabbit Anti Keap1 Polyclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology mouse anti human keap1 antibody
    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
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    Santa Cruz Biotechnology polyclonal primary antibody against 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
    Polyclonal Primary Antibody Against Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 81/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology goat anti keap1 polyclonal antibody e20
    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
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    Santa Cruz Biotechnology polyclonal antibody against mouse 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
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    Santa Cruz Biotechnology mouse anti kelch like ech associated protein 1
    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
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    Santa Cruz Biotechnology kelch like ech associated protein 1
    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
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    Santa Cruz Biotechnology mouse anti keap 1
    Effects of Tanshinone IIA on NOX4 and Nrf2/ARE signaling axis in lung tissues of silicosis rats. ( A ) Relative mRNA expression of NOX4, Nrf2, <t>Keap-1,</t> HO-1 and NQO-1 in lung tissues determined by RT-PCR analyses. ( B ) Representative bands of NOX4, Nrf2 in the cytoplasm and nucleus, Keap-1, HO-1 and NQO-1 as examined by western blotting; ( C ) Relative protein levels of NOX4, Nrf2, Keap-1, HO-1 and NQO-1 in lung tissues. Data are presented as the mean ± standard deviarion of three repeat experiments, n=6. *P
    Mouse Anti Keap 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse anti keap 1 - by Bioz Stars, 2020-02
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    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

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

    Journal: Journal of Translational Medicine

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

    doi: 10.1186/1479-5876-12-50

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

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

    Techniques: Western Blot, Expressing

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

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

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

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

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

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

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

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

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

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

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

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

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

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

    Techniques:

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

    Journal: Cell Death and Differentiation

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

    doi: 10.1038/cdd.2014.152

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

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

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

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

    Journal: Cell Death and Differentiation

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

    doi: 10.1038/cdd.2014.152

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

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

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

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M110.145441

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

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

    Techniques:

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M110.145441

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

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

    Techniques: Western Blot, Transfection, Expressing

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

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

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

    doi: 10.1155/2015/187265

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

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

    Techniques: Expressing

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

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

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

    doi: 10.1155/2015/187265

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

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

    Techniques: Expressing

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

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

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

    doi: 10.1155/2015/187265

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

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

    Techniques:

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

    Journal: EBioMedicine

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

    doi: 10.1016/j.ebiom.2015.11.045

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

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

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

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

    Journal: EBioMedicine

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

    doi: 10.1016/j.ebiom.2015.11.045

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

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

    Techniques: Expressing, Real-time Polymerase Chain Reaction

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

    Journal: EBioMedicine

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

    doi: 10.1016/j.ebiom.2015.11.045

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

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

    Techniques: Expressing, Labeling, FACS

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

    Journal: EBioMedicine

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

    doi: 10.1016/j.ebiom.2015.11.045

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

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

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

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

    Journal: Antioxidants & Redox Signaling

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

    doi: 10.1089/ars.2014.5856

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

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

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

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

    Journal: Antioxidants & Redox Signaling

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

    doi: 10.1089/ars.2014.5856

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

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

    Techniques: Expressing

    Acute proteasome stress co-opts SQSTM1 onto protein aggregates, and induces de novo SQSTM1 expression. ( A, B ) Immunofluorescence analysis of SQSTM1 and ubiquitin accumulation in MM lines upon treatment with bortezomib (Btz). Nuclei are stained blue with DAPI. Scale bars: 10 µm. ( A ) SQSTM1 in MM.1S cells left untreated (left) or treated for 1 h with 1 µM Btz (right) (n = 5 independent experiments). ( B ) SQSTM1 and ubiquitin in MM.1S cells treated with Btz (as in A ). ( C ) Co-immunoprecipitation (IP) of polyubiquitinated proteins with SQSTM1. MM.1S cells were treated with Btz (as in A ), prior to IP of SQSTM1, and the association of ubiquitinated proteins and KEAP1 with SQSTM1 assessed by immunoblot (n ≥ 3 ). ( D ) Changes of selected proteins of the SQSTM1 interactome upon treatment with Btz (as in A ) as determined by SILAC LC-MS/MS in MM.1S cells (more proteins listed in Table 1 ). ( E ) Quantitative RT-PCR analysis of transcripts encoding the indicated autophagy receptors in MM lines treated with 1 µM Btz for 4 h. mRNA amounts were normalized by histone H3 and expressed relative to untreated controls (average induction ±s.e.m.; n = 3). ( F ) Immunoblot analysis of SQSTM1 and LC3 in the indicated MM lines treated with 1 µM Btz for 8 h (representative blot, n = 3). ( G ) Immunoblot analysis of SQSTM1 in MM.1S cells treated with 1 µM Btz for 8 h in the presence or absence of 10 µg/ml cycloheximide (CHX).

    Journal: Autophagy

    Article Title: A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells

    doi: 10.1080/15548627.2015.1052928

    Figure Lengend Snippet: Acute proteasome stress co-opts SQSTM1 onto protein aggregates, and induces de novo SQSTM1 expression. ( A, B ) Immunofluorescence analysis of SQSTM1 and ubiquitin accumulation in MM lines upon treatment with bortezomib (Btz). Nuclei are stained blue with DAPI. Scale bars: 10 µm. ( A ) SQSTM1 in MM.1S cells left untreated (left) or treated for 1 h with 1 µM Btz (right) (n = 5 independent experiments). ( B ) SQSTM1 and ubiquitin in MM.1S cells treated with Btz (as in A ). ( C ) Co-immunoprecipitation (IP) of polyubiquitinated proteins with SQSTM1. MM.1S cells were treated with Btz (as in A ), prior to IP of SQSTM1, and the association of ubiquitinated proteins and KEAP1 with SQSTM1 assessed by immunoblot (n ≥ 3 ). ( D ) Changes of selected proteins of the SQSTM1 interactome upon treatment with Btz (as in A ) as determined by SILAC LC-MS/MS in MM.1S cells (more proteins listed in Table 1 ). ( E ) Quantitative RT-PCR analysis of transcripts encoding the indicated autophagy receptors in MM lines treated with 1 µM Btz for 4 h. mRNA amounts were normalized by histone H3 and expressed relative to untreated controls (average induction ±s.e.m.; n = 3). ( F ) Immunoblot analysis of SQSTM1 and LC3 in the indicated MM lines treated with 1 µM Btz for 8 h (representative blot, n = 3). ( G ) Immunoblot analysis of SQSTM1 in MM.1S cells treated with 1 µM Btz for 8 h in the presence or absence of 10 µg/ml cycloheximide (CHX).

    Article Snippet: The following Abs were used: guinea pig anti-SQSTM1 C-terminal polyclonal Ab (1:1000 dilution; ProGen, GP62-C); rabbit anti-SQSTM1 (1:1000; Sigma-Aldrich, P0067); rabbit anti-MAP1LC3 polyclonal Ab (1:500; Novus Biologicals, NB100–220); rabbit anti-ATG7 (1:1000; Sigma-Aldrich, A2856); anti-ACTB/β actin (1:2000; Sigma-Aldrich, A5441); mouse anti-TUBA4A/α tubulin (1:5000; Sigma-Aldrich, T6074); rabbit anti-P4HB (1:1000, polyclonal antibody; kind gift of Ineke Braakman, Utrecht, NL); goat anti-KEAP1 (1:200; Santa Cruz Biotechnologies, sc-15246); mouse anti-ERP44 (1:1000; monoclonal Ab (36C9), kind gift of Roberto Sitia, Milano, Italy); rabbit anti-PRDX4 (1:1000; AbFrontier, PA0009); mouse anti-Ub monoclonal Ab (P4D1; 1:500; Santa Cruz Biotechnology, sc-8017).

    Techniques: Expressing, Immunofluorescence, Staining, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Quantitative RT-PCR

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep44769

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

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

    Techniques: Expressing, Immunohistochemistry, Staining

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep44769

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

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

    Techniques: Expressing, Immunofluorescence, Staining

    p62/SQSTM1 phosphorylation at Ser351 and Keap1 downregulation in sodium arsenite-treated human normal and HGPS fibroblasts A) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. Intracellular ROS were detected after incubation with CM-H2DCFDA and flow cytometry. The results are mean + sd. of three independent experiments. The ROS level in untreated normal fibroblasts was defined as 1.0. B) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. 5 μg of cytoplasmic extracts were loaded on 10% SDS-PAGE and expression of Keap1, p62/SQSTM1, phosphorylated p62/SQSTM1 at Ser351 were analyzed by Western blotting. β-actin is shown as a loading control of each sample.

    Journal: Cellular signalling

    Article Title: Nuclear Lamins and Progerin Are Dispensable for Antioxidant Nrf2 Response to Arsenic and Cadmium

    doi: 10.1016/j.cellsig.2017.02.012

    Figure Lengend Snippet: p62/SQSTM1 phosphorylation at Ser351 and Keap1 downregulation in sodium arsenite-treated human normal and HGPS fibroblasts A) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. Intracellular ROS were detected after incubation with CM-H2DCFDA and flow cytometry. The results are mean + sd. of three independent experiments. The ROS level in untreated normal fibroblasts was defined as 1.0. B) Human normal (GM07492) and HGPS (AG11498) fibroblasts were treated with 10 μM sodium arsenite for 24 hr. 5 μg of cytoplasmic extracts were loaded on 10% SDS-PAGE and expression of Keap1, p62/SQSTM1, phosphorylated p62/SQSTM1 at Ser351 were analyzed by Western blotting. β-actin is shown as a loading control of each sample.

    Article Snippet: The primary antibodies used in this study are lamin A/C (4C11, #4777, mouse monoclonal), lamin B1 (#9087, rabbit polyclonal), and lamin B2 (#9622, rabbit polyclonal) from Cell Signaling Technology; Nrf2 (sc-13032x), HMOX1 (sc-7695, sc-136960), NQO-1 (sc-32793), histone H1 (sc-8030), progerin (sc-81611) from Santa Cruz Biotechnology, Keap1 (Millipore, clone 144), p62/SQSTM1 (BD Transduction Laboratories), Phospho-Ser351 p62/SQSTM1 (Medical and Biological Laboratories).

    Techniques: Incubation, Flow Cytometry, Cytometry, SDS Page, Expressing, Western Blot

    SQSTM1 accumulation activates the SQSTM1-KEAP1-NFE2L2 axis that reduces ROS production in EBV-infected monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) KEAP1 and NFE2L2 expression by western blot, (b) NFE2L2 localization by IFA and (c) CAT and GSR expression by western blot. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of KEAP1:ACTB, NFE2L2:ACTB, CAT:ACTB, GSR:ACTB of 3 different experiments. SQSTM1 staining is shown in red; bars: 10 mm. Differentiating monocytes exposed to EBV were silenced for si SQSTM1 with specific siRNA or scrambled siRNA and cultured for 5 days with CSF2 and IL4 before analysing (d) NFE2L2 localization by IFA and (e) SQSTM1, CAT and GSR expression by western blot. NFE2L2 staining is shown in red; bars: 10 mm. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of SQSTM1:ACTB, CAT:ACTB, GSR:ACTB of 3 different experiments. * P value

    Journal: Autophagy

    Article Title: EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation

    doi: 10.1080/15548627.2018.1536530

    Figure Lengend Snippet: SQSTM1 accumulation activates the SQSTM1-KEAP1-NFE2L2 axis that reduces ROS production in EBV-infected monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) KEAP1 and NFE2L2 expression by western blot, (b) NFE2L2 localization by IFA and (c) CAT and GSR expression by western blot. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of KEAP1:ACTB, NFE2L2:ACTB, CAT:ACTB, GSR:ACTB of 3 different experiments. SQSTM1 staining is shown in red; bars: 10 mm. Differentiating monocytes exposed to EBV were silenced for si SQSTM1 with specific siRNA or scrambled siRNA and cultured for 5 days with CSF2 and IL4 before analysing (d) NFE2L2 localization by IFA and (e) SQSTM1, CAT and GSR expression by western blot. NFE2L2 staining is shown in red; bars: 10 mm. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of SQSTM1:ACTB, CAT:ACTB, GSR:ACTB of 3 different experiments. * P value

    Article Snippet: The following primary antibodies were used in western blotting analysis: mouse monoclonal anti-EA-D (1:1000; Millipore, MAB8186), rabbit polyclonal anti-PARP1 (1:500; Cell Signaling Technology, 9542), rabbit polyclonal anti-LC3B (1:1000; Novus Biologicals, NB100-2220SS), mouse monoclonal anti-SQSTM1 (1:500; BD Transduction Laboratories, 610,883), rabbit polyclonal anti-ATG5 (1:500; Cell Signaling Technology, 2630), rabbit polyclonal anti-RAB7 (1:100; Santa Cruz Biotechnology, sc-10,767; no longer available), rabbit polyclonal anti-BECN1 (1:500; Cell Signaling Technology, 3738), mouse monoclonal anti-STAT3 (1:1000; BD Transduction Laboratories, 610,189), mouse monoclonal anti-phospho-STAT3 (p-Tyr705, 1:100; Santa Cruz Biotechnology Inc., sc-8059), mouse monoclonal anti-phospho-STAT3 (p-Ser727, 1:50; BD Transduction Laboratories, 612,543), mouse monoclonal anti-KEAP1 (1:100; Santa Cruz Biotechnology, sc-365,626), mouse monoclonal anti-NFE2L2/NRF2 (1:100; Santa Cruz Biotechnology, sc-81,342), mouse monoclonal anti-GSR (glutathione reductase) (1:100; Santa Cruz Biotechnology, sc-133,245), mouse monoclonal anti-CAT (catalase) (1:100; Santa Cruz Biotechnology, sc-271,803), mouse monoclonal anti-NRF1 (1:100; Santa Cruz Biotechnology, sc-28,379), mouse monoclonal anti-TFAM (1:100; Santa Cruz Biotechnology, sc-166,965).

    Techniques: Infection, Cell Culture, Expressing, Western Blot, Immunofluorescence, 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

    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

    Effects of Tanshinone IIA on NOX4 and Nrf2/ARE signaling axis in lung tissues of silicosis rats. ( A ) Relative mRNA expression of NOX4, Nrf2, Keap-1, HO-1 and NQO-1 in lung tissues determined by RT-PCR analyses. ( B ) Representative bands of NOX4, Nrf2 in the cytoplasm and nucleus, Keap-1, HO-1 and NQO-1 as examined by western blotting; ( C ) Relative protein levels of NOX4, Nrf2, Keap-1, HO-1 and NQO-1 in lung tissues. Data are presented as the mean ± standard deviarion of three repeat experiments, n=6. *P

    Journal: Drug Design, Development and Therapy

    Article Title: The Protective Role of Tanshinone IIA in Silicosis Rat Model via TGF-β1/Smad Signaling Suppression, NOX4 Inhibition and Nrf2/ARE Signaling Activation

    doi: 10.2147/DDDT.S230572

    Figure Lengend Snippet: Effects of Tanshinone IIA on NOX4 and Nrf2/ARE signaling axis in lung tissues of silicosis rats. ( A ) Relative mRNA expression of NOX4, Nrf2, Keap-1, HO-1 and NQO-1 in lung tissues determined by RT-PCR analyses. ( B ) Representative bands of NOX4, Nrf2 in the cytoplasm and nucleus, Keap-1, HO-1 and NQO-1 as examined by western blotting; ( C ) Relative protein levels of NOX4, Nrf2, Keap-1, HO-1 and NQO-1 in lung tissues. Data are presented as the mean ± standard deviarion of three repeat experiments, n=6. *P

    Article Snippet: After blocking with 5% non-fat milk made with Tris-buffered saline (containing 0.1% Tween 20), the membranes were incubated with the following specific primary antibodies at 4°C overnight: rabbit anti-TGF-β1 (1:1000, abcam; cat. no. ab-92486), rabbit anti-Smad3 (1:1000, abcam; cat. no. ab-40854), rabbit anti-p-Smad3 (S208) (1:1000, abcam; cat. no. ab138659), rabbit anti-Smad2/3 (1:200; Boster Biological Technology; cat. no. BA1395), rabbit anti-p-Smad2 (S465+S467)/p-Smad3 (S423+S425) (1:500; Wanlei Biological Technology, Ltd; cat. no. WL02305), rabbit anti-Smad7 (1:1000, abcam; cat. no. ab-216428), rabbit anti-NOX-4 (1:200; Boster Biological Technology; cat. no. BM4135), mouse anti-Nrf2 (1:400, Santa Cruz; cat. no. sc-81342), mouse anti Keap-1 (1:200; Santa Cruz; cat. no. sc-514914), mouse anti HO-1 (1:200; Santa Cruz; cat. no. sc-136960), mouse anti NQO-1 (1:200; Santa Cruz, Inc.; cat. no. sc-32793), rabbit β-actin (1:500; Wanlei Biological Technology, Ltd; cat. no. WL01372).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot