Structured Review

Santa Cruz Biotechnology antibody against keap1
ERK5 controls <t>Keap1</t> mRNA expression. A) 10 7 Jurkat-TAg cells were transfected with 5 μg of the empty pcDNA vector, ERK5 or a pSUPER Neo vector containing a small hairpin RNA for ERK5 (shERK5). Forty-eight hours later mRNA expression was analyzed by qPCR and presented as the % of mRNA compared to cells transfected with the control vector. B) Protein expression of cells transfected in (A). C) 10 7 Jurkat-TAg cells were transfected with 5 μg of the empty pSUPER Neo vector or with the vector encoding for the shERK5. Protein expression was analyzed by WB at different times after transfection. The data represent means ± SD; *p
Antibody Against Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Price from $9.99 to $1999.99
antibody against keap1 - by Bioz Stars, 2020-02
86/100 stars

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Images

1) Product Images from "Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS) Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS) Production"

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

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2015.11.045

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

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

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

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

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

Techniques Used: Expressing, Labeling, FACS

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

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

Related Articles

Nucleic Acid Electrophoresis:

Article Title: Hypoxia-Responsive MicroRNA-101 Promotes Angiogenesis via Heme Oxygenase-1/Vascular Endothelial Growth Factor Axis by Targeting Cullin 3
Article Snippet: Cell lysates were incubated with antibodies against Keap1 (Santa Cruz Biotechnology), Nrf2, HIF-1α (Novus Biologicals), and HSP90 (Santa Cruz Biotechnology), and immune complexes were collected by centrifugation after incubation with an antibody against protein G-Sepharose (Millipore). .. Immunoprecipitates were analyzed by sodium dodecyl sulphate polyacryl amide gel electrophoresis, followed by Western blot analysis using the indicated antibodies.

In Vivo:

Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿
Article Snippet: Cells were grown on 35-mm glass-bottom dishes (In Vivo Scientific) for live-cell imaging. .. 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).

Stable Transfection:

Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿
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). .. All images were taken with the Zeiss Observer.Z1 microscope using the Slidebook 4.2.0.11 computer program (Intelligent Imaging Innovations, Inc.).

Synthesized:

Article Title: Low Molecular Seleno-Aminopolysaccharides Protect the Intestinal Mucosal Barrier of Rats under Weaning Stress
Article Snippet: The antibody against Keap1 was acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). .. Our laboratory synthesized the low molecular seleno-aminopolysaccharide (LSA).

Multiple Displacement Amplification:

Article Title: Low Molecular Seleno-Aminopolysaccharides Protect the Intestinal Mucosal Barrier of Rats under Weaning Stress
Article Snippet: NO, TNOS, SOD, GSH-Px, CAT, and MDA were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). .. The antibody against Keap1 was acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Protease Inhibitor:

Article Title: Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS) Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS) Production
Article Snippet: The complete protease inhibitor cocktail (Complete EDTA-free) and the phosphatase inhibitor cocktail (PhosSTOP) were from Roche. .. The antibody against KEAP1 and MEF2 (E-17) were from Santa Cruz Biotechnology.

Lysis:

Article Title: Low Molecular Seleno-Aminopolysaccharides Protect the Intestinal Mucosal Barrier of Rats under Weaning Stress
Article Snippet: 2000 DNA Marker, RNA loading buffer, DNA 6×loading buffer, RIPA Lysis buffer, PMSF, BCA kit, BeyoECL Star kit, 5×loading buffer, BSA, non-fat dried milk, TEMED were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). .. The antibody against Keap1 was acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Live Cell Imaging:

Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿
Article Snippet: Cells were grown on 35-mm glass-bottom dishes (In Vivo Scientific) for live-cell imaging. .. 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).

Centrifugation:

Article Title: Hypoxia-Responsive MicroRNA-101 Promotes Angiogenesis via Heme Oxygenase-1/Vascular Endothelial Growth Factor Axis by Targeting Cullin 3
Article Snippet: .. Cell lysates were incubated with antibodies against Keap1 (Santa Cruz Biotechnology), Nrf2, HIF-1α (Novus Biologicals), and HSP90 (Santa Cruz Biotechnology), and immune complexes were collected by centrifugation after incubation with an antibody against protein G-Sepharose (Millipore). .. Immunoprecipitates were analyzed by sodium dodecyl sulphate polyacryl amide gel electrophoresis, followed by Western blot analysis using the indicated antibodies.

Microscopy:

Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿
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). .. All images were taken with the Zeiss Observer.Z1 microscope using the Slidebook 4.2.0.11 computer program (Intelligent Imaging Innovations, Inc.).

Immunoprecipitation:

Article Title: Hypoxia-Responsive MicroRNA-101 Promotes Angiogenesis via Heme Oxygenase-1/Vascular Endothelial Growth Factor Axis by Targeting Cullin 3
Article Snippet: Paragraph title: Immunoprecipitation ... Cell lysates were incubated with antibodies against Keap1 (Santa Cruz Biotechnology), Nrf2, HIF-1α (Novus Biologicals), and HSP90 (Santa Cruz Biotechnology), and immune complexes were collected by centrifugation after incubation with an antibody against protein G-Sepharose (Millipore).

Incubation:

Article Title: Hypoxia-Responsive MicroRNA-101 Promotes Angiogenesis via Heme Oxygenase-1/Vascular Endothelial Growth Factor Axis by Targeting Cullin 3
Article Snippet: .. Cell lysates were incubated with antibodies against Keap1 (Santa Cruz Biotechnology), Nrf2, HIF-1α (Novus Biologicals), and HSP90 (Santa Cruz Biotechnology), and immune complexes were collected by centrifugation after incubation with an antibody against protein G-Sepharose (Millipore). .. Immunoprecipitates were analyzed by sodium dodecyl sulphate polyacryl amide gel electrophoresis, followed by Western blot analysis using the indicated antibodies.

Imaging:

Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿
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). .. All images were taken with the Zeiss Observer.Z1 microscope using the Slidebook 4.2.0.11 computer program (Intelligent Imaging Innovations, Inc.).

BIA-KA:

Article Title: Low Molecular Seleno-Aminopolysaccharides Protect the Intestinal Mucosal Barrier of Rats under Weaning Stress
Article Snippet: 2000 DNA Marker, RNA loading buffer, DNA 6×loading buffer, RIPA Lysis buffer, PMSF, BCA kit, BeyoECL Star kit, 5×loading buffer, BSA, non-fat dried milk, TEMED were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). .. The antibody against Keap1 was acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Marker:

Article Title: Low Molecular Seleno-Aminopolysaccharides Protect the Intestinal Mucosal Barrier of Rats under Weaning Stress
Article Snippet: 2000 DNA Marker, RNA loading buffer, DNA 6×loading buffer, RIPA Lysis buffer, PMSF, BCA kit, BeyoECL Star kit, 5×loading buffer, BSA, non-fat dried milk, TEMED were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). .. The antibody against Keap1 was acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Western Blot:

Article Title: Hypoxia-Responsive MicroRNA-101 Promotes Angiogenesis via Heme Oxygenase-1/Vascular Endothelial Growth Factor Axis by Targeting Cullin 3
Article Snippet: Cell lysates were incubated with antibodies against Keap1 (Santa Cruz Biotechnology), Nrf2, HIF-1α (Novus Biologicals), and HSP90 (Santa Cruz Biotechnology), and immune complexes were collected by centrifugation after incubation with an antibody against protein G-Sepharose (Millipore). .. Immunoprecipitates were analyzed by sodium dodecyl sulphate polyacryl amide gel electrophoresis, followed by Western blot analysis using the indicated antibodies.

Chloramphenicol Acetyltransferase Assay:

Article Title: Low Molecular Seleno-Aminopolysaccharides Protect the Intestinal Mucosal Barrier of Rats under Weaning Stress
Article Snippet: NO, TNOS, SOD, GSH-Px, CAT, and MDA were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). .. The antibody against Keap1 was acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Immunofluorescence:

Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿
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). .. All images were taken with the Zeiss Observer.Z1 microscope using the Slidebook 4.2.0.11 computer program (Intelligent Imaging Innovations, Inc.).

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  • 82
    Santa Cruz Biotechnology antibodies against keap1
    The interaction between p62 and <t>Keap1</t> 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
    Antibodies Against Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 82/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against keap1/product/Santa Cruz Biotechnology
    Average 82 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    antibodies against keap1 - by Bioz Stars, 2020-02
    82/100 stars
      Buy from Supplier

    91
    Santa Cruz Biotechnology mouse antibody against keap1
    <t>Keap1,</t> MCM3, and MCM-BP form a ternary complex. ( a ) Strep-Keap1 and FLAG-MCM3 pulldown experiments from Sf9 cells co-infected with baculoviruses expressing mouse MCM-BP together with WT or interaction deficient mutant MCM3 and Keap1 as indicated. Top panels show the Western blots of indicated proteins, bottom panel the blotted membranes that were stained with colloidal gold total protein stain. 1/300th of the starting extracts (‘input’) and 1/6th of the pulldown samples was loaded on each lane. See Supplementary Fig. S6 for full-length blots. ( b ) Strep-Keap1 - FLAG-MCM3 tandem affinity purification experiment from Sf9 cells co-infected with baculoviruses expressing all six mouse MCM2-7 subunits, Keap1, and MCM-BP. Coomassie brilliant blue stained SDS-PAGE gel on the left shows eluted material from both affinity purification steps, and unbound material from the FLAG affinity step in the middle lane. Resulting complexes were further resolved by Superose 6 size exclusion chromatography, the fractions of which are shown on right gel; co-elution of molecular weight markers is indicated at the bottom. The identity of protein bands was verified by mass spectrometry.
    Mouse Antibody Against Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse antibody against keap1/product/Santa Cruz Biotechnology
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mouse antibody against keap1 - by Bioz Stars, 2020-02
    91/100 stars
      Buy from Supplier

    Image Search Results


    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:

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

    Journal: Antioxidants & Redox Signaling

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

    doi: 10.1089/ars.2014.5856

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

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

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

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

    Journal: Antioxidants & Redox Signaling

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

    doi: 10.1089/ars.2014.5856

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

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

    Techniques: Expressing

    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

    Keap1, MCM3, and MCM-BP form a ternary complex. ( a 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 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: Affinity Purification, Infection, Expressing, Staining, 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 ). 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 for images of full-length blots. ( f 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 ). 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 for images of full-length blots. ( f 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

    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 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 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: Staining, SDS Page, Infection, Expressing, 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 for full-length blots. ( b 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 for full-length blots. ( b 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: Ligation, Proximity Ligation Assay, Staining, Confocal Microscopy, Negative Control, Standard Deviation