Structured Review

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 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 95 stars, based on 1 article reviews
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1) Product Images from "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"

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

Journal: Cancer Science

doi: 10.1111/cas.13963

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
Figure Legend 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

Techniques Used: Expressing, Immunoprecipitation, Western Blot, Over Expression

Related Articles

Nucleic Acid Electrophoresis:

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‐associa
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. .. The content of vascular endothelial growth factor (VEGF) was measured with a VEGF ELISA kit (R & D Systems, Minneapolis, MI, USA) with total protein normalization.

Immunoprecipitation:

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‐associa
Article Snippet: Paragraph title: Quantitative reverse transcription polymerase chain reaction (qRT‐PCR), western blot analysis, enzyme‐linked immunosorbent assay (ELISA) and immunoprecipitation assay ... 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.

Quantitative RT-PCR:

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‐associa
Article Snippet: Paragraph title: Quantitative reverse transcription polymerase chain reaction (qRT‐PCR), western blot analysis, enzyme‐linked immunosorbent assay (ELISA) and immunoprecipitation assay ... 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.

Enzyme-linked Immunosorbent Assay:

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‐associa
Article Snippet: Paragraph title: Quantitative reverse transcription polymerase chain reaction (qRT‐PCR), western blot analysis, enzyme‐linked immunosorbent assay (ELISA) and immunoprecipitation assay ... 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.

Reverse Transcription Polymerase Chain Reaction:

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‐associa
Article Snippet: Paragraph title: Quantitative reverse transcription polymerase chain reaction (qRT‐PCR), western blot analysis, enzyme‐linked immunosorbent assay (ELISA) and immunoprecipitation assay ... 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.

Polymerase Chain Reaction:

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‐associa
Article Snippet: The resulting cDNA was used for PCR in triplicate, and data collection was performed using a Smart Cycler 2 PCR system (Cepheid, Sunnyvale, CA, USA). .. 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.

Western Blot:

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‐associa
Article Snippet: Paragraph title: Quantitative reverse transcription polymerase chain reaction (qRT‐PCR), western blot analysis, enzyme‐linked immunosorbent assay (ELISA) and immunoprecipitation assay ... 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.

SDS Page:

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‐associa
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. .. The content of vascular endothelial growth factor (VEGF) was measured with a VEGF ELISA kit (R & D Systems, Minneapolis, MI, USA) with total protein normalization.

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  • 94
    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: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    95
    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 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human keap1/product/Santa Cruz Biotechnology
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti human keap1 - by Bioz Stars, 2020-04
    95/100 stars
      Buy from Supplier

    86
    Santa Cruz Biotechnology anti keap1 2h5
    PTMA binds and orchestrates with TRIM 21 to regulate Nrf2 expression through <t>p62/Keap1</t> signaling. A, Immunoprecipitation study for the interaction between endogenous TRIM 21 and PTMA . Total protein lysate from BFTC 905 cells was immunoprecipitated either with PTMA or IgG (as a control), then immunoblotted with TRIM 21. B, Western blotting for TRIM 21, PTMA , and Nrf2 in several bladder cancer cells. Numeric in red indicates the ratio of protein of interest‐to‐β‐actin. C‐F, Western blotting for TRIM 21 and Nrf2 expression while knocking down or overexpression of the PTMA gene in the indicated cells. G, Western blotting for heme oxygenase‐1 ( HMOX 1) and superoxide dismutase‐2 ( SOD 2) expression in J82 cells with ectopic expression of WT PTMA and ∆ NLS PTMA . IgG, immunoglobulin G; Keap1, Kelch‐like ECH‐associated protein 1; Nrf2, nuclear factor erythroid 2‐related factor 2; PTMA , prothymosin‐α; TRIM 21, tripartite motif‐containing protein 21
    Anti Keap1 2h5, 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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    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

    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