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  • 98
    Santa Cruz Biotechnology anti keap1
    Schematic of SILAC-based proteomic mapping of <t>KEAP1</t> modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.
    Anti Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 98/100, based on 209 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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).
    Goat Anti Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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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    Santa Cruz Biotechnology anti keap1 polyclonal antibody
    Liver expression of Nrf2, <t>Keap1</t> and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.
    Anti Keap1 Polyclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti keap1 e20 antibodies
    Liver expression of Nrf2, <t>Keap1</t> and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.
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    Santa Cruz Biotechnology keap1 sc 365626 antibodies
    Liver expression of Nrf2, <t>Keap1</t> and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.
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    Santa Cruz Biotechnology goat polyclonal anti keap1 antibody
    Liver expression of Nrf2, <t>Keap1</t> and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.
    Goat Polyclonal Anti Keap1 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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 rabbit anti keap1 polyclonal antibody
    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: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti keap1 h 190
    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 keap1 e20 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 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 keap1 sc 33569 antibodies
    PTMA binds and orchestrates with TRIM 21 to regulate Nrf2 expression through <t>p62/Keap1</t> signaling. A, Immunoprecipitation study for the interaction between endogenous TRIM 21 and PTMA . Total protein lysate from BFTC 905 cells was immunoprecipitated either with PTMA or IgG (as a control), then immunoblotted with TRIM 21. B, Western blotting for TRIM 21, PTMA , and Nrf2 in several bladder cancer cells. Numeric in red indicates the ratio of protein of interest‐to‐β‐actin. C‐F, Western blotting for TRIM 21 and Nrf2 expression while knocking down or overexpression of the PTMA gene in the indicated cells. G, Western blotting for heme oxygenase‐1 ( HMOX 1) and superoxide dismutase‐2 ( SOD 2) expression in J82 cells with ectopic expression of WT PTMA and ∆ NLS PTMA . IgG, immunoglobulin G; Keap1, Kelch‐like ECH‐associated protein 1; Nrf2, nuclear factor erythroid 2‐related factor 2; PTMA , prothymosin‐α; TRIM 21, tripartite motif‐containing protein 21
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    Santa Cruz Biotechnology primary antibodies 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
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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
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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
    Polyclonal Antibody Against Mouse Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti nrf2 antibody
    Proposed <t>Nrf2-ARE</t> signaling pathway. Nrf2 is expressed constitutively in the cell and translocates directly to the nucleus following its synthesis. Following transactivation of its genes, Nrf2 is targeted for degradation by Keap1 in the nucleus, a
    Anti Nrf2 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 504 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti rbx1
    Overexpression of Nrf2 up-regulates endogenous and transfected Cul3 and <t>Rbx1</t> gene expression. A , Western analysis of Flp-in T-REx 293 (293) cells or 293/FRT/FLAG-Nrf2 (FRT/FLAG-Nrf2) cells expressing tetracycline-induced FLAG-tagged Nrf2 were incubated with 4 μg/ml tetracycline (TET) for the indicated times. Cells were harvested, lysed, and probed with anti-FLAG, anti-Cul3, and anti-Rbx1. Anti-β-actin was used as loading control. Densitometry measurements of bands were quantitated and shown in graph blots below. B and C , Cul3 and Rbx1 gene expression was analyzed by real time-PCR. 293 cells or FRT/FLAG-Nrf2 cells were seeded in a monolayer and treated with 4 μg/ml tetracycline for indicated time points. RNA was extracted, converted to cDNA. 50 ng of cDNA was analyzed using primers and probes specific for Cul3 and Rbx1 mRNA. Tetracycline-induced Nrf2 expression was also confirmed using specific primers for exogenous Nrf2. NQO1 and INrf2 were used as positive control, respectively. GusB primers and probes were used as internal control. D , 293 cells or FRT/FLAG-Nrf2 cells were co-transfected with Cul3 or Rbx1 ARE plasmids and the internal control plasmid pRL-TK. Twenty-four hours after transfection, the cells were treated with 4 μg/ml tetracycline for 8 or 16 h. pGL2 vector was used as negative control. The cells were harvested and analyzed for luciferase activity. The data shown are mean ± S.D. of three independent transfection experiments.
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    Santa Cruz Biotechnology mouse anti kelch like ech associated protein 1
    Overexpression of Nrf2 up-regulates endogenous and transfected Cul3 and <t>Rbx1</t> gene expression. A , Western analysis of Flp-in T-REx 293 (293) cells or 293/FRT/FLAG-Nrf2 (FRT/FLAG-Nrf2) cells expressing tetracycline-induced FLAG-tagged Nrf2 were incubated with 4 μg/ml tetracycline (TET) for the indicated times. Cells were harvested, lysed, and probed with anti-FLAG, anti-Cul3, and anti-Rbx1. Anti-β-actin was used as loading control. Densitometry measurements of bands were quantitated and shown in graph blots below. B and C , Cul3 and Rbx1 gene expression was analyzed by real time-PCR. 293 cells or FRT/FLAG-Nrf2 cells were seeded in a monolayer and treated with 4 μg/ml tetracycline for indicated time points. RNA was extracted, converted to cDNA. 50 ng of cDNA was analyzed using primers and probes specific for Cul3 and Rbx1 mRNA. Tetracycline-induced Nrf2 expression was also confirmed using specific primers for exogenous Nrf2. NQO1 and INrf2 were used as positive control, respectively. GusB primers and probes were used as internal control. D , 293 cells or FRT/FLAG-Nrf2 cells were co-transfected with Cul3 or Rbx1 ARE plasmids and the internal control plasmid pRL-TK. Twenty-four hours after transfection, the cells were treated with 4 μg/ml tetracycline for 8 or 16 h. pGL2 vector was used as negative control. The cells were harvested and analyzed for luciferase activity. The data shown are mean ± S.D. of three independent transfection experiments.
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    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
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    Image Search Results


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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

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

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

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

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

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

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

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

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

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

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

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

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

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

    Techniques:

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

    Journal: Journal of Translational Medicine

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

    doi: 10.1186/1479-5876-12-50

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

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

    Techniques: Western Blot, Expressing

    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:

    Treatment with MS-275 decreases expression of KEAP1 and increases miR-200a expression in H82 cells. KEAP1 mRNA ( A ) and protein ( B ) expression levels were determined by qRT-PCR and quantitative Western blot, respectively, in H82 cells following 48-h co-treatments.

    Journal: Molecular cancer therapeutics

    Article Title: Histone deacetylase inhibition overcomes drug resistance through a miRNA-dependent mechanism

    doi: 10.1158/1535-7163.MCT-13-0418

    Figure Lengend Snippet: Treatment with MS-275 decreases expression of KEAP1 and increases miR-200a expression in H82 cells. KEAP1 mRNA ( A ) and protein ( B ) expression levels were determined by qRT-PCR and quantitative Western blot, respectively, in H82 cells following 48-h co-treatments.

    Article Snippet: The KEAP1 antibody (1:500 dilution) was purchased from Santa Cruz and β-actin was used as a loading control and for normalization.

    Techniques: Mass Spectrometry, Expressing, Quantitative RT-PCR, Western Blot

    The interaction between p62 and Keap1 and the domains that are required for the interaction. (A) Identification of the Keap1-interacting protein p62. Two stable cell lines, MDA-MB-231 and Keap1 −/− , that expressed either vector control

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: The interaction between p62 and Keap1 and the domains that are required for the interaction. (A) Identification of the Keap1-interacting protein p62. Two stable cell lines, MDA-MB-231 and Keap1 −/− , that expressed either vector control

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques: Stable Transfection, Multiple Displacement Amplification, Plasmid Preparation

    p62 decreased ubiquitination of Nrf2, leading to an increase in Nrf2 stability. (A) p62 decreased the ubiquitination of Nrf2 and increased the ubiquitination of Keap1. HEK293 cells were cotransfected with an expression vector for Nrf2, Keap1, HA-ubiquitin,

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: p62 decreased ubiquitination of Nrf2, leading to an increase in Nrf2 stability. (A) p62 decreased the ubiquitination of Nrf2 and increased the ubiquitination of Keap1. HEK293 cells were cotransfected with an expression vector for Nrf2, Keap1, HA-ubiquitin,

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques: Expressing, Plasmid Preparation

    p62 sequestered Keap1 into aggregates. (A) The cellular localization of p62, Keap1, Nrf2, and Cul3. HEK293 cells were singly transfected with an expression vector for the fluorescently tagged protein. The subcellular localization of the proteins was monitored

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: p62 sequestered Keap1 into aggregates. (A) The cellular localization of p62, Keap1, Nrf2, and Cul3. HEK293 cells were singly transfected with an expression vector for the fluorescently tagged protein. The subcellular localization of the proteins was monitored

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques: Transfection, Expressing, Plasmid Preparation

    Autophagy-defective cells sequestered Keap1 into aggregates. (A and B) Keap1 was sequestered into aggregates in primary autophagy-deficient cells. Atg5 +/+ , Atg5 −/− , Beclin1 +/+ , and Beclin +/−

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: Autophagy-defective cells sequestered Keap1 into aggregates. (A and B) Keap1 was sequestered into aggregates in primary autophagy-deficient cells. Atg5 +/+ , Atg5 −/− , Beclin1 +/+ , and Beclin +/−

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques:

    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

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.25.11.4501-4513.2005

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

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

    Techniques: Inhibition, Translocation Assay, Activation Assay

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.25.11.4501-4513.2005

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

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

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

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.25.11.4501-4513.2005

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

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

    Techniques: Construct, Fluorescence, Staining

    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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0122358

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0122358

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

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

    Techniques: Expressing, Western Blot, Quantitative RT-PCR

    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

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.R900010200

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

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

    Techniques:

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.R900010200

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

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

    Techniques: Expressing

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.R900010200

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

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

    Techniques:

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M110.121863

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

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

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

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M110.121863

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

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

    Techniques: Activity Assay

    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