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Sustained <t>Nrf2</t> nuclear translocation in the TG hearts induces gene expression of ARE-dependent antioxidants. Real-time RT-PCR determinations of Nrf2 target genes in NTG and TG mouse at 3 (A–H) and 6 months (I–P) were performed using Qiagen-mouse
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1) Product Images from "Sustained Activation of Nuclear Erythroid 2-Related Factor 2/Antioxidant Response Element Signaling Promotes Reductive Stress in the Human Mutant Protein Aggregation Cardiomyopathy in Mice"

Article Title: Sustained Activation of Nuclear Erythroid 2-Related Factor 2/Antioxidant Response Element Signaling Promotes Reductive Stress in the Human Mutant Protein Aggregation Cardiomyopathy in Mice

Journal: Antioxidants & Redox Signaling

doi: 10.1089/ars.2010.3587

Sustained Nrf2 nuclear translocation in the TG hearts induces gene expression of ARE-dependent antioxidants. Real-time RT-PCR determinations of Nrf2 target genes in NTG and TG mouse at 3 (A–H) and 6 months (I–P) were performed using Qiagen-mouse
Figure Legend Snippet: Sustained Nrf2 nuclear translocation in the TG hearts induces gene expression of ARE-dependent antioxidants. Real-time RT-PCR determinations of Nrf2 target genes in NTG and TG mouse at 3 (A–H) and 6 months (I–P) were performed using Qiagen-mouse

Techniques Used: Translocation Assay, Expressing, Quantitative RT-PCR

Schematic proposal for Nrf2 activation and mechanism for reductive stress in the mutant-protein aggregation cardiomyopathy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article
Figure Legend Snippet: Schematic proposal for Nrf2 activation and mechanism for reductive stress in the mutant-protein aggregation cardiomyopathy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article

Techniques Used: Activation Assay, Mutagenesis

Decreased ubiquitination and increased activity of Nrf2 in the TG mouse. (A) TransAM-Nrf2 activity assay: Nuclear extracts from NTG and TG mouse ( n = 6) at 6 months were incubated with the precoated antioxidant response element (ARE) oligonucleotides.
Figure Legend Snippet: Decreased ubiquitination and increased activity of Nrf2 in the TG mouse. (A) TransAM-Nrf2 activity assay: Nuclear extracts from NTG and TG mouse ( n = 6) at 6 months were incubated with the precoated antioxidant response element (ARE) oligonucleotides.

Techniques Used: Activity Assay, Incubation

Sequestration of Keap1 in to the mutant CryAB aggregates facilitate Nrf2 nuclear translocation. Immunofluorescence analysis was performed using the CryAB (Rb.ab, green) and Keap1 (SC-Goat ab, red) and merged with the nuclear staining (DRAQ1). CryAB and
Figure Legend Snippet: Sequestration of Keap1 in to the mutant CryAB aggregates facilitate Nrf2 nuclear translocation. Immunofluorescence analysis was performed using the CryAB (Rb.ab, green) and Keap1 (SC-Goat ab, red) and merged with the nuclear staining (DRAQ1). CryAB and

Techniques Used: Mutagenesis, Translocation Assay, Immunofluorescence, Staining

2) Product Images from "Disturbance of redox status enhances radiosensitivity of hepatocellular carcinoma"

Article Title: Disturbance of redox status enhances radiosensitivity of hepatocellular carcinoma

Journal: American Journal of Cancer Research

doi:

Effects of ISL on Keap-1 and Nrf-2 on HepG2 cells. A. After various times of ISL treatment, expressions of Keap-1 and Nrf-2 mRNA were measured by RT-PCR. β-Actin was used as a standard. B. After various time treatments with ISL, expressions of Keap-1 and Nrf-2 proteins were measured by western blot. C. After various time treatments with ISL, ubiquitination of Nrf2 was measured by immunoprecipitation. All data are expressed as mean ± SEM from three independent experiments. * p
Figure Legend Snippet: Effects of ISL on Keap-1 and Nrf-2 on HepG2 cells. A. After various times of ISL treatment, expressions of Keap-1 and Nrf-2 mRNA were measured by RT-PCR. β-Actin was used as a standard. B. After various time treatments with ISL, expressions of Keap-1 and Nrf-2 proteins were measured by western blot. C. After various time treatments with ISL, ubiquitination of Nrf2 was measured by immunoprecipitation. All data are expressed as mean ± SEM from three independent experiments. * p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation

3) Product Images from "Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa"

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-30562-y

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Sequencing, Binding Assay

Helix-2 insert (H2I) hairpin and its conserved ETGE sequence box are required for normal growth and minichromosome maintenance function of MCM3 in yeast. ( a ) Tetrad dissection of yeast strains that carry mcm3 alleles with the mutations in H2I motif as depicted schematically on the right (dashed lines correspond to deleted regions). For each MCM3/mcm3 diploid strain, two tetrads are shown that were grown for three days after dissection. Arrowheads indicate clones with a mutated allele. ( b ) Competitive co-growth of wild type (WT) yeast with strains carrying mcm3 - GAGA (upper panel) or mcm3 - del449 -454 (lower panel) mutant alleles. The WT and mutant strains were pre-grown separately before mixing together on day 0 and co-growing for four days. Genomic DNA from a resulting co-culture was analysed by PCR and following restriction analysis with AluI ( mcm3 - GAGA ) or XhoI ( mcm3 - del449 - 454 ), which cleave the mutant but not WT DNA fragment. ( c ) Plasmid minichromosome maintenance assay with mcm3 - GAGA and mcm3 - del449 - 454 strains. Cells were transformed with pRS416 plasmid and grown for four days without selection, determining the percentage of plasmid-carrying cells each day.
Figure Legend Snippet: Helix-2 insert (H2I) hairpin and its conserved ETGE sequence box are required for normal growth and minichromosome maintenance function of MCM3 in yeast. ( a ) Tetrad dissection of yeast strains that carry mcm3 alleles with the mutations in H2I motif as depicted schematically on the right (dashed lines correspond to deleted regions). For each MCM3/mcm3 diploid strain, two tetrads are shown that were grown for three days after dissection. Arrowheads indicate clones with a mutated allele. ( b ) Competitive co-growth of wild type (WT) yeast with strains carrying mcm3 - GAGA (upper panel) or mcm3 - del449 -454 (lower panel) mutant alleles. The WT and mutant strains were pre-grown separately before mixing together on day 0 and co-growing for four days. Genomic DNA from a resulting co-culture was analysed by PCR and following restriction analysis with AluI ( mcm3 - GAGA ) or XhoI ( mcm3 - del449 - 454 ), which cleave the mutant but not WT DNA fragment. ( c ) Plasmid minichromosome maintenance assay with mcm3 - GAGA and mcm3 - del449 - 454 strains. Cells were transformed with pRS416 plasmid and grown for four days without selection, determining the percentage of plasmid-carrying cells each day.

Techniques Used: Sequencing, Dissection, Clone Assay, Mutagenesis, Co-Culture Assay, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay, Selection

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: Ligation, Proximity Ligation Assay, Staining, Confocal Microscopy, Negative Control, Standard Deviation

4) Product Images from "KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿"

Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.05036-11

Overexpression of KPNA6 attenuates the inducible Nrf2 signaling in response to oxidative stress. (A) KPNA6 decreases the inducible Nrf2 protein level and the expression of its downstream detoxification genes without altering Keap1 protein levels. HEK293T
Figure Legend Snippet: Overexpression of KPNA6 attenuates the inducible Nrf2 signaling in response to oxidative stress. (A) KPNA6 decreases the inducible Nrf2 protein level and the expression of its downstream detoxification genes without altering Keap1 protein levels. HEK293T

Techniques Used: Over Expression, Expressing

Schematic model of Nrf2 regulation by Keap1. Keap1 is a key regulator of the Nrf2-signaling pathway and serves as a molecular switch to turn the Nrf2-mediated antioxidant response on and off. (1) Oxidative stress or chemopreventive compounds cause a conformational
Figure Legend Snippet: Schematic model of Nrf2 regulation by Keap1. Keap1 is a key regulator of the Nrf2-signaling pathway and serves as a molecular switch to turn the Nrf2-mediated antioxidant response on and off. (1) Oxidative stress or chemopreventive compounds cause a conformational

Techniques Used:

The nuclear import of Keap1 occurs at its physiological protein level and is not dependent on Nrf1 or Nrf2. (A) GFP-tagged Keap1 proteins were expressed at levels similar to the levels of endogenous Keap1 in the stable MEF cell lines. Keap1 −/−
Figure Legend Snippet: The nuclear import of Keap1 occurs at its physiological protein level and is not dependent on Nrf1 or Nrf2. (A) GFP-tagged Keap1 proteins were expressed at levels similar to the levels of endogenous Keap1 in the stable MEF cell lines. Keap1 −/−

Techniques Used:

KPNA6 modulates the antioxidant response by promoting ubiquitination and degradation of Nrf2. (A) KPNA6 is required for efficient repression of nuclear Nrf2 protein levels during the postinduction phase of the antioxidant response. MDA-MB-231 cells were
Figure Legend Snippet: KPNA6 modulates the antioxidant response by promoting ubiquitination and degradation of Nrf2. (A) KPNA6 is required for efficient repression of nuclear Nrf2 protein levels during the postinduction phase of the antioxidant response. MDA-MB-231 cells were

Techniques Used: Multiple Displacement Amplification

5) Product Images from "Seven in Absentia Homolog 2 (Siah2) Protein Is a Regulator of NF-E2-related Factor 2 (Nrf2) *Seven in Absentia Homolog 2 (Siah2) Protein Is a Regulator of NF-E2-related Factor 2 (Nrf2) * ♦"

Article Title: Seven in Absentia Homolog 2 (Siah2) Protein Is a Regulator of NF-E2-related Factor 2 (Nrf2) *Seven in Absentia Homolog 2 (Siah2) Protein Is a Regulator of NF-E2-related Factor 2 (Nrf2) * ♦

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.438762

Effect of hypoxia on Siah2 accumulation. Hep3B cells were exposed to hypoxia for 6 h. Sixty, 40, 20, and 5 μg of lysates were immunoblotted with anti-Siah2, anti-HIF-1α, anti-Nrf2, or anti-β-actin antibodies.
Figure Legend Snippet: Effect of hypoxia on Siah2 accumulation. Hep3B cells were exposed to hypoxia for 6 h. Sixty, 40, 20, and 5 μg of lysates were immunoblotted with anti-Siah2, anti-HIF-1α, anti-Nrf2, or anti-β-actin antibodies.

Techniques Used:

Isolation of Human Nrf2, Keap1, and Siah2
Figure Legend Snippet: Isolation of Human Nrf2, Keap1, and Siah2

Techniques Used: Isolation

Association of Siah2 with Nrf2. A, Hep3B cells were transfected with pcDNA3.1 containing Myc-Nrf2 , V172A , or V172A/P174A . Forty eight hours after transfection, the cells were exposed to hypoxia for 6 h in the presence of MG132 (5 μ m ). Total cell
Figure Legend Snippet: Association of Siah2 with Nrf2. A, Hep3B cells were transfected with pcDNA3.1 containing Myc-Nrf2 , V172A , or V172A/P174A . Forty eight hours after transfection, the cells were exposed to hypoxia for 6 h in the presence of MG132 (5 μ m ). Total cell

Techniques Used: Transfection

Effect of Siah2 knockdown on accumulation and ubiquitination of Nrf2(93–604) and association of Siah2 with Nrf2(93–604). A, empty pcDNA3.1 vector or pcDNA3.1 containing Myc-Nrf2 , or Myc-Nrf2 ( 93–604 ) was transfected into MOCK (GFP
Figure Legend Snippet: Effect of Siah2 knockdown on accumulation and ubiquitination of Nrf2(93–604) and association of Siah2 with Nrf2(93–604). A, empty pcDNA3.1 vector or pcDNA3.1 containing Myc-Nrf2 , or Myc-Nrf2 ( 93–604 ) was transfected into MOCK (GFP

Techniques Used: Plasmid Preparation, Transfection

Effect of Siah2 overexpression on Nrf2 accumulation. Hep3B cells were transfected with empty pcDNA4 vector ( MOCK ) or pcDNA4 containing FLAG-Siah2 . Forty eight hours after transfection, the transfected and WT Hep3B cells were grown under normoxia or hypoxia
Figure Legend Snippet: Effect of Siah2 overexpression on Nrf2 accumulation. Hep3B cells were transfected with empty pcDNA4 vector ( MOCK ) or pcDNA4 containing FLAG-Siah2 . Forty eight hours after transfection, the transfected and WT Hep3B cells were grown under normoxia or hypoxia

Techniques Used: Over Expression, Transfection, Plasmid Preparation

Determination of Siah2-binding site in Nrf2 amino acid residues. Hep3B cells were transfected with empty pcDNA3.1 vector ( MOCK ) or pcDNA3.1 containing Myc-Nrf2 , V172A , or V172A/P174A . Forty eight hours after transfection, the cells were grown under normoxia
Figure Legend Snippet: Determination of Siah2-binding site in Nrf2 amino acid residues. Hep3B cells were transfected with empty pcDNA3.1 vector ( MOCK ) or pcDNA3.1 containing Myc-Nrf2 , V172A , or V172A/P174A . Forty eight hours after transfection, the cells were grown under normoxia

Techniques Used: Binding Assay, Transfection, Plasmid Preparation

Significance of Nrf2 phosphorylation by PKC. A, total cell lysates were immunoprecipitated using anti-IgG, anti-Siah2, or anti-Keap1 antibodies and then immunoblotted using anti-Nrf2 antibody. B, lysates were immunoprecipitated using anti-IgG, anti-Nrf2,
Figure Legend Snippet: Significance of Nrf2 phosphorylation by PKC. A, total cell lysates were immunoprecipitated using anti-IgG, anti-Siah2, or anti-Keap1 antibodies and then immunoblotted using anti-Nrf2 antibody. B, lysates were immunoprecipitated using anti-IgG, anti-Nrf2,

Techniques Used: Immunoprecipitation

Effect of Siah2 knockdown on Nrf2 accumulation and transcription of Nrf2-responsive genes. A, WT Hep3B, MOCK (GFP shRNA control), or Siah2 knockdown Hep3B, HEK293 and HeLa cells were grown under normoxia or hypoxia for 6 h. Two independent Siah2 knockdown
Figure Legend Snippet: Effect of Siah2 knockdown on Nrf2 accumulation and transcription of Nrf2-responsive genes. A, WT Hep3B, MOCK (GFP shRNA control), or Siah2 knockdown Hep3B, HEK293 and HeLa cells were grown under normoxia or hypoxia for 6 h. Two independent Siah2 knockdown

Techniques Used: shRNA

6) Product Images from "Defective protein repair under methionine sulfoxide A deletion drives autophagy and ARE-dependent gene transcription"

Article Title: Defective protein repair under methionine sulfoxide A deletion drives autophagy and ARE-dependent gene transcription

Journal: Redox Biology

doi: 10.1016/j.redox.2018.04.001

Elevated Nrf2 protein expression under MsrA deficiency is due to protein stabilization rather than increased transcription. (A, B) Representative immunoblot (A) and quantification (B) for Nrf2 protein levels in whole cell lysates from MsrA-/- and WT VSMC; n = 3 biological replicates. (C) Nrf2 mRNA levels in VSMC by qRT-PCR; n = 5 biological replicates. (D) Representative immunoprecipitation of Nrf2 followed by immunoblot for ubiquitin in MsrA-/- and WT VSMC. IgG: IP with IgG, WT + MC132: IP with anti-Nrf2 in WT VSMC incubated with MG132, WCL: whole cell lysate of WT VSMC as controls. (E) Quantification of (D); n = 7 biological replicates. (F) p62 mRNA levels by qRT-PCR in aortic samples WT, MsrA-/- and MsrA-/- x Nrf2-/- mice; n = 7, 9 biological replicates. (G) Representative Immunoblots for Nrf2 and GAPDH in aortic samples from WT, MsrA-/- and MsrA-/- x Nrf2-/- mice. (H) Quantification of (G) n = 7 biological replicates. (E) * p
Figure Legend Snippet: Elevated Nrf2 protein expression under MsrA deficiency is due to protein stabilization rather than increased transcription. (A, B) Representative immunoblot (A) and quantification (B) for Nrf2 protein levels in whole cell lysates from MsrA-/- and WT VSMC; n = 3 biological replicates. (C) Nrf2 mRNA levels in VSMC by qRT-PCR; n = 5 biological replicates. (D) Representative immunoprecipitation of Nrf2 followed by immunoblot for ubiquitin in MsrA-/- and WT VSMC. IgG: IP with IgG, WT + MC132: IP with anti-Nrf2 in WT VSMC incubated with MG132, WCL: whole cell lysate of WT VSMC as controls. (E) Quantification of (D); n = 7 biological replicates. (F) p62 mRNA levels by qRT-PCR in aortic samples WT, MsrA-/- and MsrA-/- x Nrf2-/- mice; n = 7, 9 biological replicates. (G) Representative Immunoblots for Nrf2 and GAPDH in aortic samples from WT, MsrA-/- and MsrA-/- x Nrf2-/- mice. (H) Quantification of (G) n = 7 biological replicates. (E) * p

Techniques Used: Expressing, Quantitative RT-PCR, Immunoprecipitation, Incubation, Mouse Assay, Western Blot

7) Product Images from "Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis"

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

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2014.49

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

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

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

Techniques Used: Stable Transfection, Expressing, Trypan Blue Exclusion Assay

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

Techniques Used: Stable Transfection, Expressing, Trypan Blue Exclusion Assay

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

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

8) Product Images from "A mechanism for the suppression of homologous recombination in G1 cells"

Article Title: A mechanism for the suppression of homologous recombination in G1 cells

Journal: Nature

doi: 10.1038/nature16142

a, Schematic representation of human 53BP1 gene organization and targeting sites of sgRNAs used. Boxes indicate exons (E: yellow, coding sequence; brown, untranslated regions (UTRs)). The indels introduced by CRISPR/Cas9 and their respective frequencies are indicated. b , Wild-type (WT) and 53BP1Δ and U2OS cells were mock- or X-irradiated (10 Gy) before being processed for 53BP1 fluorescence microscopy. DAPI was used to stain DNA and trace the outline of the nucleus. c , Wild-type (WT) and 53BP1Δ U2OS cells were processed for 53BP1 immunoblotting. Tubulin was used as a loading control. d , Wild-type (WT) and 53BP1Δ U2OS cells either synchronized in G1 following a double-thymidine block and release or asynchronously dividing (ASN), were irradiated (2 Gy) and processed for γ-H2AX, PALB2, BRCA2 and BRCA1 immunofluorescence. The micrographs relating to BRCA1 and BRCA2 staining in G1 are found in . e , Wild-type (WT) and 53BP1Δ U2OS cells synchronized in G1 after release from a double-thymidine block were irradiated (20 Gy) and processed for γ-H2AX, BRCA1 and BRCA2 immunofluorescence. On the left are representative micrographs for the G1-arrested cells and the quantitation of the full experiment is shown on the right (mean ± s.d., N =3). Fig. 1a
Figure Legend Snippet: a, Schematic representation of human 53BP1 gene organization and targeting sites of sgRNAs used. Boxes indicate exons (E: yellow, coding sequence; brown, untranslated regions (UTRs)). The indels introduced by CRISPR/Cas9 and their respective frequencies are indicated. b , Wild-type (WT) and 53BP1Δ and U2OS cells were mock- or X-irradiated (10 Gy) before being processed for 53BP1 fluorescence microscopy. DAPI was used to stain DNA and trace the outline of the nucleus. c , Wild-type (WT) and 53BP1Δ U2OS cells were processed for 53BP1 immunoblotting. Tubulin was used as a loading control. d , Wild-type (WT) and 53BP1Δ U2OS cells either synchronized in G1 following a double-thymidine block and release or asynchronously dividing (ASN), were irradiated (2 Gy) and processed for γ-H2AX, PALB2, BRCA2 and BRCA1 immunofluorescence. The micrographs relating to BRCA1 and BRCA2 staining in G1 are found in . e , Wild-type (WT) and 53BP1Δ U2OS cells synchronized in G1 after release from a double-thymidine block were irradiated (20 Gy) and processed for γ-H2AX, BRCA1 and BRCA2 immunofluorescence. On the left are representative micrographs for the G1-arrested cells and the quantitation of the full experiment is shown on the right (mean ± s.d., N =3). Fig. 1a

Techniques Used: Sequencing, CRISPR, Irradiation, Fluorescence, Microscopy, Staining, Blocking Assay, Immunofluorescence, Quantitation Assay

9) Product Images from "Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis"

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

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2014.49

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

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

10) Product Images from "Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa"

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-30562-y

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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 .
Figure Legend 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 .

Techniques Used: Sequencing

11) Product Images from "Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity"

Article Title: Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.150

Beneficial effects of PTP1B deficiency on the induction of Nrf2-mediated antioxidant response and survival signaling in the liver. PTP1B +/+ and PTP1B −/− mice were injected with 300 mg/kg APAP or saline for 3 or 6 h. ( a ) Western blot analysis of Nrf2 in nuclear extracts and HO-1, phospho (p)-JNK and JNK in total liver extracts. ( b ) GPx, HO-1, GCL-M, GCL-C and NQO1 mRNA levels determined by quantitative real-time polymerase chain reaction (qRT-PCR) at 3 and 6 h after APAP injection. * P
Figure Legend Snippet: Beneficial effects of PTP1B deficiency on the induction of Nrf2-mediated antioxidant response and survival signaling in the liver. PTP1B +/+ and PTP1B −/− mice were injected with 300 mg/kg APAP or saline for 3 or 6 h. ( a ) Western blot analysis of Nrf2 in nuclear extracts and HO-1, phospho (p)-JNK and JNK in total liver extracts. ( b ) GPx, HO-1, GCL-M, GCL-C and NQO1 mRNA levels determined by quantitative real-time polymerase chain reaction (qRT-PCR) at 3 and 6 h after APAP injection. * P

Techniques Used: Mouse Assay, Injection, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

12) Product Images from "Dysfunctional KEAP1-NRF2 Interaction in Non-Small-Cell Lung Cancer"

Article Title: Dysfunctional KEAP1-NRF2 Interaction in Non-Small-Cell Lung Cancer

Journal: PLoS Medicine

doi: 10.1371/journal.pmed.0030420

Status of KEAP1 and NRF2 Is Altered in Cancer Cells (A) Immunoblot showing increased nuclear localization of NRF2 in nuclear extracts (NE) from cancer cells. Cancer cells showed lower levels of KEAP1 (~69 kDa) and higher levels of NRF2 (~110 kDa) in total protein lysates (TP). NIVT and KIVT indicate NRF2 and KEAP1 in vitro transcribed/translated product, respectively. (B and C) Quantification of NRF2 and KEAP1 protein in immunoblots. For band densitometry, bands in nuclear extract blot (B) were normalized to Lamin B1, and those in total protein (C) were normalized to GAPDH. (D) Heat map showing relative expression of  KEAP1, NRF2,  and NRF2-dependent genes by real-time RT-PCR. Raw data for the heat maps are presented in   Table S5 .
Figure Legend Snippet: Status of KEAP1 and NRF2 Is Altered in Cancer Cells (A) Immunoblot showing increased nuclear localization of NRF2 in nuclear extracts (NE) from cancer cells. Cancer cells showed lower levels of KEAP1 (~69 kDa) and higher levels of NRF2 (~110 kDa) in total protein lysates (TP). NIVT and KIVT indicate NRF2 and KEAP1 in vitro transcribed/translated product, respectively. (B and C) Quantification of NRF2 and KEAP1 protein in immunoblots. For band densitometry, bands in nuclear extract blot (B) were normalized to Lamin B1, and those in total protein (C) were normalized to GAPDH. (D) Heat map showing relative expression of KEAP1, NRF2, and NRF2-dependent genes by real-time RT-PCR. Raw data for the heat maps are presented in Table S5 .

Techniques Used: In Vitro, Western Blot, Expressing, Quantitative RT-PCR

13) Product Images from "Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa"

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-30562-y

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

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

Helix-2 insert (H2I) hairpin and its conserved ETGE sequence box are required for normal growth and minichromosome maintenance function of MCM3 in yeast. ( a ) Tetrad dissection of yeast strains that carry mcm3 alleles with the mutations in H2I motif as depicted schematically on the right (dashed lines correspond to deleted regions). For each MCM3/mcm3 diploid strain, two tetrads are shown that were grown for three days after dissection. Arrowheads indicate clones with a mutated allele. ( b ) Competitive co-growth of wild type (WT) yeast with strains carrying mcm3 - GAGA (upper panel) or mcm3 - del449 -454 (lower panel) mutant alleles. The WT and mutant strains were pre-grown separately before mixing together on day 0 and co-growing for four days. Genomic DNA from a resulting co-culture was analysed by PCR and following restriction analysis with AluI ( mcm3 - GAGA ) or XhoI ( mcm3 - del449 - 454 ), which cleave the mutant but not WT DNA fragment. ( c ) Plasmid minichromosome maintenance assay with mcm3 - GAGA and mcm3 - del449 - 454 strains. Cells were transformed with pRS416 plasmid and grown for four days without selection, determining the percentage of plasmid-carrying cells each day.
Figure Legend Snippet: Helix-2 insert (H2I) hairpin and its conserved ETGE sequence box are required for normal growth and minichromosome maintenance function of MCM3 in yeast. ( a ) Tetrad dissection of yeast strains that carry mcm3 alleles with the mutations in H2I motif as depicted schematically on the right (dashed lines correspond to deleted regions). For each MCM3/mcm3 diploid strain, two tetrads are shown that were grown for three days after dissection. Arrowheads indicate clones with a mutated allele. ( b ) Competitive co-growth of wild type (WT) yeast with strains carrying mcm3 - GAGA (upper panel) or mcm3 - del449 -454 (lower panel) mutant alleles. The WT and mutant strains were pre-grown separately before mixing together on day 0 and co-growing for four days. Genomic DNA from a resulting co-culture was analysed by PCR and following restriction analysis with AluI ( mcm3 - GAGA ) or XhoI ( mcm3 - del449 - 454 ), which cleave the mutant but not WT DNA fragment. ( c ) Plasmid minichromosome maintenance assay with mcm3 - GAGA and mcm3 - del449 - 454 strains. Cells were transformed with pRS416 plasmid and grown for four days without selection, determining the percentage of plasmid-carrying cells each day.

Techniques Used: Sequencing, Dissection, Clone Assay, Mutagenesis, Co-Culture Assay, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay, Selection

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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

14) Product Images from "Keap1–MCM3 interaction is a potential coordinator of molecular machineries of antioxidant response and genomic DNA replication in metazoa"

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-30562-y

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: Ligation, Proximity Ligation Assay, Staining, Confocal Microscopy, Negative Control, Standard Deviation

15) Product Images from "Proteasome Dysfunction Activates Autophagy and the Keap1-Nrf2 Pathway *"

Article Title: Proteasome Dysfunction Activates Autophagy and the Keap1-Nrf2 Pathway *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.580357

Autophagic degradation of ubiquitin-positive aggregates accumulated due to impaired proteasome activity. A , mice of the indicated genotypes were subjected to intraperitoneal injection of leupeptin at P30. One hour after the injection, liver homogenates
Figure Legend Snippet: Autophagic degradation of ubiquitin-positive aggregates accumulated due to impaired proteasome activity. A , mice of the indicated genotypes were subjected to intraperitoneal injection of leupeptin at P30. One hour after the injection, liver homogenates

Techniques Used: Activity Assay, Mouse Assay, Injection

16) Product Images from "Impaired Nuclear Nrf2 Translocation Undermines the Oxidative Stress Response in Friedreich Ataxia"

Article Title: Impaired Nuclear Nrf2 Translocation Undermines the Oxidative Stress Response in Friedreich Ataxia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004253

Frataxin-depleted SKNAS cells. A. Anti-frataxin antibody produced strong mitochondrial labeling in control SKNAS cells (a) and severely reduced labeling in SKNAS cells treated with frataxin-targeted shRNA (shRNAFra SKNAS cells) (b). B. Residual frataxin mRNA in patient fibroblasts and shRNAFra SKNAS cells compared to control cells. C. Actin and Nrf2 in control (a, c, e, g) and shRNAFra (b, d, f, h) SKNAS cells under basal conditions (a, b, c, d) or after t BHQ treatment (e, f, g, h). Actin staining with phalloidin (Phal) shows disorganization of the actin stress fibers in shRNAFra SKNAS cells (a) compared to control SKNAS cells (b), in keeping with the results in fibroblasts. Nrf2 labeling produced similar abundant staining of the cytoplasm of both control SKNAS cells (c) and shRNAFra SKNAS cells (d). Nuclear translocation of Nrf2 occurs in control SKNAS cells (e, g) but not in shRNAFra SKNAS cells. D. Induction of Phase II antioxidants in SKNAS cells using oligomycin (a) or t BHQ (b). Significant differences were noted (** p
Figure Legend Snippet: Frataxin-depleted SKNAS cells. A. Anti-frataxin antibody produced strong mitochondrial labeling in control SKNAS cells (a) and severely reduced labeling in SKNAS cells treated with frataxin-targeted shRNA (shRNAFra SKNAS cells) (b). B. Residual frataxin mRNA in patient fibroblasts and shRNAFra SKNAS cells compared to control cells. C. Actin and Nrf2 in control (a, c, e, g) and shRNAFra (b, d, f, h) SKNAS cells under basal conditions (a, b, c, d) or after t BHQ treatment (e, f, g, h). Actin staining with phalloidin (Phal) shows disorganization of the actin stress fibers in shRNAFra SKNAS cells (a) compared to control SKNAS cells (b), in keeping with the results in fibroblasts. Nrf2 labeling produced similar abundant staining of the cytoplasm of both control SKNAS cells (c) and shRNAFra SKNAS cells (d). Nuclear translocation of Nrf2 occurs in control SKNAS cells (e, g) but not in shRNAFra SKNAS cells. D. Induction of Phase II antioxidants in SKNAS cells using oligomycin (a) or t BHQ (b). Significant differences were noted (** p

Techniques Used: Produced, Labeling, shRNA, Staining, Translocation Assay

Effect of frataxin depletion on oxidative stress resistance, oxidative properties, and actin-Nrf2 signaling pathway status in cultured skin fibroblasts. A. Sensitivity of control and patient cells to oligomycin (30 µM for 4 days). Cells were harvested at 24-hour intervals and live cells were counted. The proportion of surviving cells was significantly different between controls and patients (ANOVA p
Figure Legend Snippet: Effect of frataxin depletion on oxidative stress resistance, oxidative properties, and actin-Nrf2 signaling pathway status in cultured skin fibroblasts. A. Sensitivity of control and patient cells to oligomycin (30 µM for 4 days). Cells were harvested at 24-hour intervals and live cells were counted. The proportion of surviving cells was significantly different between controls and patients (ANOVA p

Techniques Used: Cell Culture

Diagram of the actin-Nrf2 signaling pathway in control and patient cells under basal conditions and during oxidative stress. Under basal conditions, the Nrf2-Keap1 complex, or a sub-pool of it attached to the mitochondrial outer membrane by the PGAM5 protein, is bound to the actin stress filament network of control cells. B. In frataxin-depleted cells, characterized by abnormal iron handling, the actin-Nrf2 signaling pathway is profoundly altered by the need to cope with elevated H 2 O 2 levels. Removing H 2 O 2 with the catalase mimetic Euk134 corrects these alterations. C. Treating control cells with oligomycin or t BHQ results in major oxidative stress that destabilizes the actin-Nrf2-Keap1 complex, leading to Nrf2 release and phosphorylation. Nuclear translocation of Nrf2 results in the recruitment of the co-activator(s) needed for Phase II antioxidant transcription. D. In frataxin-depleted cells, the oxidative insult induced by oligomycin (endogenous) or t BHQ (exogenous) cannot be counterbalanced by the induction of Phase II antioxidants, so that the cells are extremely sensitive to oxidation. Again, Euk134 treatment restores the actin-Nrf2 signaling pathway, allowing transcription of Phase II antioxidants.
Figure Legend Snippet: Diagram of the actin-Nrf2 signaling pathway in control and patient cells under basal conditions and during oxidative stress. Under basal conditions, the Nrf2-Keap1 complex, or a sub-pool of it attached to the mitochondrial outer membrane by the PGAM5 protein, is bound to the actin stress filament network of control cells. B. In frataxin-depleted cells, characterized by abnormal iron handling, the actin-Nrf2 signaling pathway is profoundly altered by the need to cope with elevated H 2 O 2 levels. Removing H 2 O 2 with the catalase mimetic Euk134 corrects these alterations. C. Treating control cells with oligomycin or t BHQ results in major oxidative stress that destabilizes the actin-Nrf2-Keap1 complex, leading to Nrf2 release and phosphorylation. Nuclear translocation of Nrf2 results in the recruitment of the co-activator(s) needed for Phase II antioxidant transcription. D. In frataxin-depleted cells, the oxidative insult induced by oligomycin (endogenous) or t BHQ (exogenous) cannot be counterbalanced by the induction of Phase II antioxidants, so that the cells are extremely sensitive to oxidation. Again, Euk134 treatment restores the actin-Nrf2 signaling pathway, allowing transcription of Phase II antioxidants.

Techniques Used: Translocation Assay

17) Product Images from "A mechanism for the suppression of homologous recombination in G1 cells"

Article Title: A mechanism for the suppression of homologous recombination in G1 cells

Journal: Nature

doi: 10.1038/nature16142

a, Schematic representation of human 53BP1 gene organization and targeting sites of sgRNAs used. Boxes indicate exons (E: yellow, coding sequence; brown, untranslated regions (UTRs)). The indels introduced by CRISPR/Cas9 and their respective frequencies are indicated. b , Wild-type (WT) and 53BP1Δ and U2OS cells were mock- or X-irradiated (10 Gy) before being processed for 53BP1 fluorescence microscopy. DAPI was used to stain DNA and trace the outline of the nucleus. c , Wild-type (WT) and 53BP1Δ U2OS cells were processed for 53BP1 immunoblotting. Tubulin was used as a loading control. d , Wild-type (WT) and 53BP1Δ U2OS cells either synchronized in G1 following a double-thymidine block and release or asynchronously dividing (ASN), were irradiated (2 Gy) and processed for γ-H2AX, PALB2, BRCA2 and BRCA1 immunofluorescence. The micrographs relating to BRCA1 and BRCA2 staining in G1 are found in . e , Wild-type (WT) and 53BP1Δ U2OS cells synchronized in G1 after release from a double-thymidine block were irradiated (20 Gy) and processed for γ-H2AX, BRCA1 and BRCA2 immunofluorescence. On the left are representative micrographs for the G1-arrested cells and the quantitation of the full experiment is shown on the right (mean ± s.d., N =3). Fig. 1a
Figure Legend Snippet: a, Schematic representation of human 53BP1 gene organization and targeting sites of sgRNAs used. Boxes indicate exons (E: yellow, coding sequence; brown, untranslated regions (UTRs)). The indels introduced by CRISPR/Cas9 and their respective frequencies are indicated. b , Wild-type (WT) and 53BP1Δ and U2OS cells were mock- or X-irradiated (10 Gy) before being processed for 53BP1 fluorescence microscopy. DAPI was used to stain DNA and trace the outline of the nucleus. c , Wild-type (WT) and 53BP1Δ U2OS cells were processed for 53BP1 immunoblotting. Tubulin was used as a loading control. d , Wild-type (WT) and 53BP1Δ U2OS cells either synchronized in G1 following a double-thymidine block and release or asynchronously dividing (ASN), were irradiated (2 Gy) and processed for γ-H2AX, PALB2, BRCA2 and BRCA1 immunofluorescence. The micrographs relating to BRCA1 and BRCA2 staining in G1 are found in . e , Wild-type (WT) and 53BP1Δ U2OS cells synchronized in G1 after release from a double-thymidine block were irradiated (20 Gy) and processed for γ-H2AX, BRCA1 and BRCA2 immunofluorescence. On the left are representative micrographs for the G1-arrested cells and the quantitation of the full experiment is shown on the right (mean ± s.d., N =3). Fig. 1a

Techniques Used: Sequencing, CRISPR, Irradiation, Fluorescence, Microscopy, Staining, Blocking Assay, Immunofluorescence, Quantitation Assay

a, Site-specific chemical ubiquitylation of HA-PALB2 (1-103) at residue 20 (PALB2-K C 20-Ub) and 45 (PALB2-K C 45-Ub) was carried out by dichloroacetone linking. The resulting ubiquitylated PALB2 polypeptides along with their unmodified counterparts were subjected to pulldown with a fusion of MBP with the coiled-coil domain of BRCA1 (MBP-BRCA1-CC). I, input; PD, pulldown. Asterisk (*) indicates a non-specific band. b , Wild-type and KEAP1Δ 293T cells were treated with cycloheximide (CHX) for the indicated time and then processed for NRF2 and KEAP1 immunoblotting. Actin levels were also determined as a loading control. c , Immunoprecipitation (IP) of USP11 from extracts prepared from 293T cells that were or were not treated with camptothecin (CPT; 200 nM). IP with normal IgG was performed as a control. d , U2OS DR-GFP cells were transfected with the indicated siRNAs. 24 h post-transfection, cells were further transfected with the indicated siRNA-resistant USP11 expression vectors (WT=wild type; CS= C318S and CA= C318A catalytically-dead mutants) or an empty vector (EV), with or without an I-SceI expression vector. The percentage of GFP-positive cells was determined 48 h post-plasmid transfection for each condition and was normalized to the I-SceI + non-targeting (siCTRL) condition (mean ± s.d., N =3). e , Schematic representation of human USP11 (top) and KEAP1 (bottom) gene organization and targeting sites of sgRNAs (as described in ) used to generate the USP11Δ and USP11Δ / KEAP1Δ 293T cells. The indels introduced by the CRISPR/Cas9 and their respective frequencies are indicated. The USP11 knockout was created first and subsequently used to make the USP11Δ / KEAP1Δ double mutant. f , Immunoprecipitation (IP) of PALB2 from extracts prepared from 293T cells transfected with the indicated siRNA and with or without CPT (200 nM) treatment. IP with normal IgG was performed as a control. Extended Data Figure 1a
Figure Legend Snippet: a, Site-specific chemical ubiquitylation of HA-PALB2 (1-103) at residue 20 (PALB2-K C 20-Ub) and 45 (PALB2-K C 45-Ub) was carried out by dichloroacetone linking. The resulting ubiquitylated PALB2 polypeptides along with their unmodified counterparts were subjected to pulldown with a fusion of MBP with the coiled-coil domain of BRCA1 (MBP-BRCA1-CC). I, input; PD, pulldown. Asterisk (*) indicates a non-specific band. b , Wild-type and KEAP1Δ 293T cells were treated with cycloheximide (CHX) for the indicated time and then processed for NRF2 and KEAP1 immunoblotting. Actin levels were also determined as a loading control. c , Immunoprecipitation (IP) of USP11 from extracts prepared from 293T cells that were or were not treated with camptothecin (CPT; 200 nM). IP with normal IgG was performed as a control. d , U2OS DR-GFP cells were transfected with the indicated siRNAs. 24 h post-transfection, cells were further transfected with the indicated siRNA-resistant USP11 expression vectors (WT=wild type; CS= C318S and CA= C318A catalytically-dead mutants) or an empty vector (EV), with or without an I-SceI expression vector. The percentage of GFP-positive cells was determined 48 h post-plasmid transfection for each condition and was normalized to the I-SceI + non-targeting (siCTRL) condition (mean ± s.d., N =3). e , Schematic representation of human USP11 (top) and KEAP1 (bottom) gene organization and targeting sites of sgRNAs (as described in ) used to generate the USP11Δ and USP11Δ / KEAP1Δ 293T cells. The indels introduced by the CRISPR/Cas9 and their respective frequencies are indicated. The USP11 knockout was created first and subsequently used to make the USP11Δ / KEAP1Δ double mutant. f , Immunoprecipitation (IP) of PALB2 from extracts prepared from 293T cells transfected with the indicated siRNA and with or without CPT (200 nM) treatment. IP with normal IgG was performed as a control. Extended Data Figure 1a

Techniques Used: Immunoprecipitation, Cycling Probe Technology, Transfection, Expressing, Plasmid Preparation, CRISPR, Knock-Out, Mutagenesis

a, Quantitation of gene targeting efficiency at the LMNA locus in asynchronously dividing U2OS cells transfected with increasing amount of donor template and with (black) or without (grey) gRNAs. Gene targeting events were detected by flow cytometry (mean ± s.d., N≥ 3). b , Quantitation of gene targeting efficiency at the LMNA locus in asynchronously dividing cells transfected with the indicated siRNA. Gene targeting events were detected by flow cytometry (mean ± s.d., N =3). c , Gene targeting efficiency at the PML locus measured by flow cytometry in G1-arrested 53BP1Δ U2OS cells expressing the CtIP-T847E mutant and co-transfected with the indicated siRNA or a PALB2-KR expression construct (mean ± s.d., N =3). d , Representative FACS profiles showing the gating for 1N DNA content cells and the detection of mClover-positive cells in the LMNA gene targeting assay in asynchronous (ASN) or G1-arrested 53BP1Δ U2OS cells expressing the CtIP-T847E mutant and co-transfected with the indicated siRNA or a PALB2-KR expression construct. e , Gene targeting efficiency at the LMNA locus measured by flow cytometry in G1-arrested parental (WT) and 53BP1Δ U2OS cells transfected with KEAP1 siRNA and expressing the CtIP-T847E mutant (mean ± s.d., N =3). f , Gene targeting efficiency at the LMNA locus measured by flow cytometry in G1-arrested parental (WT) and 53BP1Δ U2OS cells transfected with the indicated siRNA and expressing either wild-type (WT) or the CtIP-T847E mutant (mean ± s.d., N =3).
Figure Legend Snippet: a, Quantitation of gene targeting efficiency at the LMNA locus in asynchronously dividing U2OS cells transfected with increasing amount of donor template and with (black) or without (grey) gRNAs. Gene targeting events were detected by flow cytometry (mean ± s.d., N≥ 3). b , Quantitation of gene targeting efficiency at the LMNA locus in asynchronously dividing cells transfected with the indicated siRNA. Gene targeting events were detected by flow cytometry (mean ± s.d., N =3). c , Gene targeting efficiency at the PML locus measured by flow cytometry in G1-arrested 53BP1Δ U2OS cells expressing the CtIP-T847E mutant and co-transfected with the indicated siRNA or a PALB2-KR expression construct (mean ± s.d., N =3). d , Representative FACS profiles showing the gating for 1N DNA content cells and the detection of mClover-positive cells in the LMNA gene targeting assay in asynchronous (ASN) or G1-arrested 53BP1Δ U2OS cells expressing the CtIP-T847E mutant and co-transfected with the indicated siRNA or a PALB2-KR expression construct. e , Gene targeting efficiency at the LMNA locus measured by flow cytometry in G1-arrested parental (WT) and 53BP1Δ U2OS cells transfected with KEAP1 siRNA and expressing the CtIP-T847E mutant (mean ± s.d., N =3). f , Gene targeting efficiency at the LMNA locus measured by flow cytometry in G1-arrested parental (WT) and 53BP1Δ U2OS cells transfected with the indicated siRNA and expressing either wild-type (WT) or the CtIP-T847E mutant (mean ± s.d., N =3).

Techniques Used: Quantitation Assay, Transfection, Flow Cytometry, Cytometry, Expressing, Mutagenesis, Construct, FACS

USP11 opposes the activity of CRL3-KEAP1 a, Normal IgG or PALB2 immunoprecipitation (IP) of extracts derived from CPT-treated 293T cells of the indicated genotypes transfected with GFP-USP11 constructs. EV, empty vector; WT, wild type; CS, C318S. b , Clonogenic survival assays of 293T cells of the indicated genotypes treated with olaparib (mean ± s.d., N≥ 3). c , Normal IgG or PALB2 IP of extracts derived from CPT-treated 293T cells of the indicated genotypes. d , Immunoblots of deubiquitylation reactions containing ubiquitylated HA-tagged PALB2 (1-103) and increasing concentrations of GST-USP11 or its C318S (CS) mutant. USP2 was used as a control. e , Cell cycle-synchronized U2OS cells were irradiated (20 Gy dose) and processed for immunoblotting. f , Immunoblots of extracts from irradiated U2OS cells transfected with the indicated siRNAs. g , Fluorescence micrographs of G1-synchronized and irradiated (20 Gy) 53BP1Δ U2OS cells transfected with the indicated siRNAs. The percentage of cells with more than 5 γ-H2AX-colocalizing BRCA2 foci is indicated (mean ± s.d., N =3).
Figure Legend Snippet: USP11 opposes the activity of CRL3-KEAP1 a, Normal IgG or PALB2 immunoprecipitation (IP) of extracts derived from CPT-treated 293T cells of the indicated genotypes transfected with GFP-USP11 constructs. EV, empty vector; WT, wild type; CS, C318S. b , Clonogenic survival assays of 293T cells of the indicated genotypes treated with olaparib (mean ± s.d., N≥ 3). c , Normal IgG or PALB2 IP of extracts derived from CPT-treated 293T cells of the indicated genotypes. d , Immunoblots of deubiquitylation reactions containing ubiquitylated HA-tagged PALB2 (1-103) and increasing concentrations of GST-USP11 or its C318S (CS) mutant. USP2 was used as a control. e , Cell cycle-synchronized U2OS cells were irradiated (20 Gy dose) and processed for immunoblotting. f , Immunoblots of extracts from irradiated U2OS cells transfected with the indicated siRNAs. g , Fluorescence micrographs of G1-synchronized and irradiated (20 Gy) 53BP1Δ U2OS cells transfected with the indicated siRNAs. The percentage of cells with more than 5 γ-H2AX-colocalizing BRCA2 foci is indicated (mean ± s.d., N =3).

Techniques Used: Activity Assay, Immunoprecipitation, Derivative Assay, Cycling Probe Technology, Transfection, Construct, Plasmid Preparation, Western Blot, Mutagenesis, Irradiation, Fluorescence

Ubiquitylation of PALB2 prevents BRCA1-PALB2 interaction a, Sequence of the PALB2 N-terminus and mutants. b , GFP IP of extracts derived from G1- or S-phase synchronized 293T cells expressing the indicated GFP-PALB2 proteins. c , In vitro ubiquitylation of the indicated HA-tagged PALB2 proteins by CRL3-KEAP1. d , Pulldown assay of ubiquitylated HA-PALB2 (1-103) incubated with MBP or MBP-BRCA1-CC. I: input, PD: pulldown, FT: flow-through. The asterisk denotes a fragment of HA-PALB2 competent for BRCA1 binding.
Figure Legend Snippet: Ubiquitylation of PALB2 prevents BRCA1-PALB2 interaction a, Sequence of the PALB2 N-terminus and mutants. b , GFP IP of extracts derived from G1- or S-phase synchronized 293T cells expressing the indicated GFP-PALB2 proteins. c , In vitro ubiquitylation of the indicated HA-tagged PALB2 proteins by CRL3-KEAP1. d , Pulldown assay of ubiquitylated HA-PALB2 (1-103) incubated with MBP or MBP-BRCA1-CC. I: input, PD: pulldown, FT: flow-through. The asterisk denotes a fragment of HA-PALB2 competent for BRCA1 binding.

Techniques Used: Sequencing, Derivative Assay, Expressing, In Vitro, Incubation, Flow Cytometry, Binding Assay

Reactivation of HR in G1 a, Quantitation of wild type (WT) and 53BP1Δ U2OS cells co-transfected with non-targeting (CTRL) or KEAP1 siRNAs and vectors expressing WT CtIP or the T847E (TE) mutant that were synchronized in G1, irradiated (2 Gy) and processed for γ-H2AX and RAD51 immunofluorescence (mean ± s.d., N =3). b , Representative micrographs from a. c , Schematic of the gene targeting assay. d , Gene targeting efficiency at the LMNA locus in asynchronously dividing (ASN) and G1-arrested U2OS cells (mean ± s.d., N =3). e , Gene targeting at the LMNA locus in G1-arrested cells transfected with the indicated siRNA or a PALB2-KR expression vector (mean ± s.d., N =3). f , Model of the cell-cycle regulation of HR.
Figure Legend Snippet: Reactivation of HR in G1 a, Quantitation of wild type (WT) and 53BP1Δ U2OS cells co-transfected with non-targeting (CTRL) or KEAP1 siRNAs and vectors expressing WT CtIP or the T847E (TE) mutant that were synchronized in G1, irradiated (2 Gy) and processed for γ-H2AX and RAD51 immunofluorescence (mean ± s.d., N =3). b , Representative micrographs from a. c , Schematic of the gene targeting assay. d , Gene targeting efficiency at the LMNA locus in asynchronously dividing (ASN) and G1-arrested U2OS cells (mean ± s.d., N =3). e , Gene targeting at the LMNA locus in G1-arrested cells transfected with the indicated siRNA or a PALB2-KR expression vector (mean ± s.d., N =3). f , Model of the cell-cycle regulation of HR.

Techniques Used: Quantitation Assay, Transfection, Expressing, Mutagenesis, Irradiation, Immunofluorescence, Plasmid Preparation

a, HEK293 Flp-In T-REX cells expressing doxycycline (DOX)-inducible His 6 -Ub were transfected with the indicated siRNAs. Cells were processed for Ni-NTA pull-down. b , 293T cells transfected with an siRNA targeting USP11 and a Flag-PALB2 expression vector were processed for Flag immunoprecipitation followed by mass spectrometry. Representative MS/MS spectra of tryptic diglycine (diG)-PALB2 peptides identified are shown (K16, top; K43, bottom). c , Schematic of the LacO /LacR chromatin-targeting system and the in vivo quantification of ubiquitylated PALB2. d , Representative micrographs of U2OS 256 cells transfected with the indicated mCherry-LacR-PALB2 vectors. Cells were processed for FK2 immunofluorescence. EV, empty vector. Scale bar = 5 μm. e , Quantification of U2OS 256 cells transfected with the indicated mCherry-LacR-PALB2 vectors. Cells were processed for quantification of FK2 fluorescence at the LacO focus. Each circle represents a cell analyzed from 3 independent experiments and the bar is at the median. Cells were also stained with a Cyclin A antibody to determine cell cycle position. Statistical significance was determined by a Kruskall-Wallis test (***, P
Figure Legend Snippet: a, HEK293 Flp-In T-REX cells expressing doxycycline (DOX)-inducible His 6 -Ub were transfected with the indicated siRNAs. Cells were processed for Ni-NTA pull-down. b , 293T cells transfected with an siRNA targeting USP11 and a Flag-PALB2 expression vector were processed for Flag immunoprecipitation followed by mass spectrometry. Representative MS/MS spectra of tryptic diglycine (diG)-PALB2 peptides identified are shown (K16, top; K43, bottom). c , Schematic of the LacO /LacR chromatin-targeting system and the in vivo quantification of ubiquitylated PALB2. d , Representative micrographs of U2OS 256 cells transfected with the indicated mCherry-LacR-PALB2 vectors. Cells were processed for FK2 immunofluorescence. EV, empty vector. Scale bar = 5 μm. e , Quantification of U2OS 256 cells transfected with the indicated mCherry-LacR-PALB2 vectors. Cells were processed for quantification of FK2 fluorescence at the LacO focus. Each circle represents a cell analyzed from 3 independent experiments and the bar is at the median. Cells were also stained with a Cyclin A antibody to determine cell cycle position. Statistical significance was determined by a Kruskall-Wallis test (***, P

Techniques Used: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Mass Spectrometry, In Vivo, Immunofluorescence, Fluorescence, Staining

53BP1Δ U2OS cells were transfected with the indicated siRNA, synchronized in G1 or S/G2 by release from a double-thymidine block and irradiated (20 Gy) before being processed for fluorescence microscopy. DAPI was used to trace the nuclear boundary and Cyclin A staining was used to determine cell cycle position. The percentage of cells with more than 5 γ-H2AX-colocalizing PALB2 foci is indicated as the mean ± s.d., N =3. Scale bar = 5 μm. b , Represent ative micrographs of irradiated G1-synchronized wild-type (WT) and 53BP1Δ U2OS cells transfected with the indicated siRNA and expressing wild-type (WT) CtIP. c , Representative micrographs of irradiated G1-synchronized WT U2OS cells transfected with the indicated siRNA and expressing CtIP-T847E. d , U2OS 53BP1Δ cells were synchronized in G1, supplemented with BrdU, irradiated (2 Gy) and processed for γ-H2AX and BrdU immunofluorescence. The percentage of cells with more than 5 γ-H2AX-colocalizing BrdU foci is indicated (mean ± s.d., N =3). e , Micrograph of a U2OS cell targeted with the CRISPR/mClover system showing the typical perinuclear expression pattern of Lamin A. f , Micrograph of a U2OS cell targeted with the mClover system showing an expression pattern characteristic of subnuclear PML foci. g , Timeline of the gene targeting ( LMNA ) experiment presented in . h , Timeline of the gene targeting ( LMNA or PML and Extended Data Figure 10. Fig 4d
Figure Legend Snippet: 53BP1Δ U2OS cells were transfected with the indicated siRNA, synchronized in G1 or S/G2 by release from a double-thymidine block and irradiated (20 Gy) before being processed for fluorescence microscopy. DAPI was used to trace the nuclear boundary and Cyclin A staining was used to determine cell cycle position. The percentage of cells with more than 5 γ-H2AX-colocalizing PALB2 foci is indicated as the mean ± s.d., N =3. Scale bar = 5 μm. b , Represent ative micrographs of irradiated G1-synchronized wild-type (WT) and 53BP1Δ U2OS cells transfected with the indicated siRNA and expressing wild-type (WT) CtIP. c , Representative micrographs of irradiated G1-synchronized WT U2OS cells transfected with the indicated siRNA and expressing CtIP-T847E. d , U2OS 53BP1Δ cells were synchronized in G1, supplemented with BrdU, irradiated (2 Gy) and processed for γ-H2AX and BrdU immunofluorescence. The percentage of cells with more than 5 γ-H2AX-colocalizing BrdU foci is indicated (mean ± s.d., N =3). e , Micrograph of a U2OS cell targeted with the CRISPR/mClover system showing the typical perinuclear expression pattern of Lamin A. f , Micrograph of a U2OS cell targeted with the mClover system showing an expression pattern characteristic of subnuclear PML foci. g , Timeline of the gene targeting ( LMNA ) experiment presented in . h , Timeline of the gene targeting ( LMNA or PML and Extended Data Figure 10. Fig 4d

Techniques Used: Transfection, Blocking Assay, Irradiation, Fluorescence, Microscopy, Staining, Expressing, Immunofluorescence, CRISPR

a, Schematic of the LacO /LacR chromatin-targeting system. b , U2OS 256 cells were transfected with the indicated mCherry-LacR and GFP-fusions. GFP fluorescence was measured at the site of the LacO array-localized mCherry focus. Each circle represents one cell analysed and the bar is at the median. Cells were also stained with a Cyclin A antibody to determine cell cycle position ( N =3). c , Representative micrographs of U2OS 256 cells transfected with the indicated mCherry-LacR and GFP-fusions; data is quantified in d. d , Quantification of U2OS 256 cells transfected with the indicated mCherry-LacR and GFP-fusions to tether either BRCA1 or PALB2 to the LacO array ( N =3). e , Schematic representation of PALB2 architecture and its major interacting proteins. f , Quantification of U2OS 256 cells transfected with the indicated GFP-PALB2 mutants and mCherry-LacR-BRCA1-CC. Cells were also stained with a Cyclin A antibody to determine cell cycle position ( N =3).
Figure Legend Snippet: a, Schematic of the LacO /LacR chromatin-targeting system. b , U2OS 256 cells were transfected with the indicated mCherry-LacR and GFP-fusions. GFP fluorescence was measured at the site of the LacO array-localized mCherry focus. Each circle represents one cell analysed and the bar is at the median. Cells were also stained with a Cyclin A antibody to determine cell cycle position ( N =3). c , Representative micrographs of U2OS 256 cells transfected with the indicated mCherry-LacR and GFP-fusions; data is quantified in d. d , Quantification of U2OS 256 cells transfected with the indicated mCherry-LacR and GFP-fusions to tether either BRCA1 or PALB2 to the LacO array ( N =3). e , Schematic representation of PALB2 architecture and its major interacting proteins. f , Quantification of U2OS 256 cells transfected with the indicated GFP-PALB2 mutants and mCherry-LacR-BRCA1-CC. Cells were also stained with a Cyclin A antibody to determine cell cycle position ( N =3).

Techniques Used: Transfection, Fluorescence, Staining

a, U2OS DR-GFP cells were transfected with the indicated siRNAs or left untransfected (−). 24 h post-transfection, cells were transfected with an I-SceI expression vector (circle). The percentage of GFP-positive cells was determined 48 h post-plasmid transfection for each condition and was normalized to the I-SceI + non-targeting (CTRL) condition (mean ± s.d., N =3). b , Parental 293T cells (WT) or a USP11Δ derivative were transfected with the indicated GFP-PALB2 constructs, treated with CPT and processed for GFP immunoprecipitation (IP). c , Parental 293T cells (WT) or a USP11Δ derivative were transfected with an empty vector (EV) or the indicated PALB2 expression vectors. Sensitivity of the cells to the PARP inhibitor olaparib was then determined by a clonogenic survival assay (mean ± s.d., N =3).
Figure Legend Snippet: a, U2OS DR-GFP cells were transfected with the indicated siRNAs or left untransfected (−). 24 h post-transfection, cells were transfected with an I-SceI expression vector (circle). The percentage of GFP-positive cells was determined 48 h post-plasmid transfection for each condition and was normalized to the I-SceI + non-targeting (CTRL) condition (mean ± s.d., N =3). b , Parental 293T cells (WT) or a USP11Δ derivative were transfected with the indicated GFP-PALB2 constructs, treated with CPT and processed for GFP immunoprecipitation (IP). c , Parental 293T cells (WT) or a USP11Δ derivative were transfected with an empty vector (EV) or the indicated PALB2 expression vectors. Sensitivity of the cells to the PARP inhibitor olaparib was then determined by a clonogenic survival assay (mean ± s.d., N =3).

Techniques Used: Transfection, Expressing, Plasmid Preparation, Construct, Cycling Probe Technology, Immunoprecipitation, Clonogenic Cell Survival Assay

a, U2OS cells synchronized in G1 or S/G2 were treated with cyclohexamide (CHX) and processed at the indicated time points to monitor USP11 stability. b , Immunoprecipitation (IP) of PALB2 from extracts prepared from 293T cells that were synchronized in G1 or S phase and treated or not with IR (20 Gy). c , U2OS cells were irradiated with a dose of 2 or 20 Gy and processed for USP11 immunoblotting at the indicated times post-IR. Actin was used as a loading control. d , U2OS cells, mock-treated or incubated with the ATM (KU55933; ATMi), ATR (VE-821; ATRi) or DNA-PKcs (NU7441; DNAPKi) inhibitors, were irradiated (20 Gy) and processed for USP11 and actin (loading control) immunoblotting. e , Similar experiment to d except that cells were UV-irradiated with a 50 mJ/cm 2 dose. f , U2OS cells, mock-treated or incubated with the proteasome inhibitor MG132, were irradiated (20 Gy) and processed for USP11 and actin (loading control) immunoblotting. g , U2OS cells, mock-treated or incubated with the cullin inhibitor MLN4924, were irradiated (20 Gy) and processed for USP11 and actin (loading control) immunoblotting.
Figure Legend Snippet: a, U2OS cells synchronized in G1 or S/G2 were treated with cyclohexamide (CHX) and processed at the indicated time points to monitor USP11 stability. b , Immunoprecipitation (IP) of PALB2 from extracts prepared from 293T cells that were synchronized in G1 or S phase and treated or not with IR (20 Gy). c , U2OS cells were irradiated with a dose of 2 or 20 Gy and processed for USP11 immunoblotting at the indicated times post-IR. Actin was used as a loading control. d , U2OS cells, mock-treated or incubated with the ATM (KU55933; ATMi), ATR (VE-821; ATRi) or DNA-PKcs (NU7441; DNAPKi) inhibitors, were irradiated (20 Gy) and processed for USP11 and actin (loading control) immunoblotting. e , Similar experiment to d except that cells were UV-irradiated with a 50 mJ/cm 2 dose. f , U2OS cells, mock-treated or incubated with the proteasome inhibitor MG132, were irradiated (20 Gy) and processed for USP11 and actin (loading control) immunoblotting. g , U2OS cells, mock-treated or incubated with the cullin inhibitor MLN4924, were irradiated (20 Gy) and processed for USP11 and actin (loading control) immunoblotting.

Techniques Used: Immunoprecipitation, Irradiation, Incubation

a, Representative micrographs of the experiment shown in . b , Schematic representation of human KEAP1 . a. The indels introduced by CRISPR/Cas9 and their respective frequencies are indicated. c , Immunoprecipitation (IP) of PALB2 from extracts prepared from irradiated 293T cells. IP with normal IgG was performed as a control. d , 293T cells with the indicated genotypes were transfected with the indicated HA-KEAP1 constructs, synchronized in G1 or S phases and irradiated. Cells were processed for PALB2 immunoprecipitation (IP). EV, empty vector. e , Quantification of U2OS 256 cells transfected with the indicated GFP-PALB2 mutants and mCherry-LacR-BRCA1. Cells were also stained with a Cyclin A antibody to determine cell cycle position ( N =3). f , Quantification of U2OS 256 cells transfected with GFP-PALB2 and mCherry-LacR-BRCA1-CC (WT or K1406R mutant). Cells were also stained with a Cyclin A antibody to determine cell cycle position. This panel shows that the sole lysine in the PALB2-interaction motif of BRCA1 is not involved in the cell cycle regulation of the PALB2-BRCA1 interaction. Fig. 1d
Figure Legend Snippet: a, Representative micrographs of the experiment shown in . b , Schematic representation of human KEAP1 . a. The indels introduced by CRISPR/Cas9 and their respective frequencies are indicated. c , Immunoprecipitation (IP) of PALB2 from extracts prepared from irradiated 293T cells. IP with normal IgG was performed as a control. d , 293T cells with the indicated genotypes were transfected with the indicated HA-KEAP1 constructs, synchronized in G1 or S phases and irradiated. Cells were processed for PALB2 immunoprecipitation (IP). EV, empty vector. e , Quantification of U2OS 256 cells transfected with the indicated GFP-PALB2 mutants and mCherry-LacR-BRCA1. Cells were also stained with a Cyclin A antibody to determine cell cycle position ( N =3). f , Quantification of U2OS 256 cells transfected with GFP-PALB2 and mCherry-LacR-BRCA1-CC (WT or K1406R mutant). Cells were also stained with a Cyclin A antibody to determine cell cycle position. This panel shows that the sole lysine in the PALB2-interaction motif of BRCA1 is not involved in the cell cycle regulation of the PALB2-BRCA1 interaction. Fig. 1d

Techniques Used: CRISPR, Immunoprecipitation, Irradiation, Transfection, Construct, Plasmid Preparation, Staining, Mutagenesis

18) Product Images from "Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity"

Article Title: Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.150

PTP1B modulates GSK3 β /Src-Fyn-mediated Nrf2 nuclear accumulation in APAP-treated hepatocytes. ( a ) PTP1B +/+ immortalized hepatocytes were transfected with control or GSK3 β small interfering RNAs (siRNAs) (25 nM) for 48 h, followed by stimulation with 1 mM APAP. Nuclear and cytosolic extracts were analyzed by western blot with antibodies against Nrf2, Fyn and Src. ( b , upper panel) PTP1B +/+ immortalized hepatocytes were treated with PP2 (5 μ M) for 30 min following 1 mM APAP for different periods. Nrf2 in nuclear extracts was analyzed by western blot. (Lower panel) SYF −/− MEFs were treated with 1 mM APAP for different periods and Nrf2 was analyzed in nuclear extracts by western blot. ( c ) PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with 1 mM APAP for different periods. Phosphorylated GSK3β (Tyr216) was analyzed in cytosolic extracts by western blot. Fyn and Src were analyzed in nuclear extracts. Similar results were obtained in three independent experiments
Figure Legend Snippet: PTP1B modulates GSK3 β /Src-Fyn-mediated Nrf2 nuclear accumulation in APAP-treated hepatocytes. ( a ) PTP1B +/+ immortalized hepatocytes were transfected with control or GSK3 β small interfering RNAs (siRNAs) (25 nM) for 48 h, followed by stimulation with 1 mM APAP. Nuclear and cytosolic extracts were analyzed by western blot with antibodies against Nrf2, Fyn and Src. ( b , upper panel) PTP1B +/+ immortalized hepatocytes were treated with PP2 (5 μ M) for 30 min following 1 mM APAP for different periods. Nrf2 in nuclear extracts was analyzed by western blot. (Lower panel) SYF −/− MEFs were treated with 1 mM APAP for different periods and Nrf2 was analyzed in nuclear extracts by western blot. ( c ) PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with 1 mM APAP for different periods. Phosphorylated GSK3β (Tyr216) was analyzed in cytosolic extracts by western blot. Fyn and Src were analyzed in nuclear extracts. Similar results were obtained in three independent experiments

Techniques Used: Transfection, Western Blot

19) Product Images from "Control of antioxidative response by the tumor suppressor protein PML through regulating Nrf2 activity"

Article Title: Control of antioxidative response by the tumor suppressor protein PML through regulating Nrf2 activity

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E13-11-0692

SFN-mediated Nrf2 activation is PML dependent. (A) ROS accumulation in PML knockdown HUVECs. HUVECs were transfected with a nontargeting siRNA or a PML siRNA for 72 h. Equal number of HUVECs were replated in a 96-well plate and treated with vehicle control (DMSO) or SFN at 40 μM for 1 h. The ROS level was measured according to the manufacturer's instruction. Data presented as mean ± SD from triplicates. * p
Figure Legend Snippet: SFN-mediated Nrf2 activation is PML dependent. (A) ROS accumulation in PML knockdown HUVECs. HUVECs were transfected with a nontargeting siRNA or a PML siRNA for 72 h. Equal number of HUVECs were replated in a 96-well plate and treated with vehicle control (DMSO) or SFN at 40 μM for 1 h. The ROS level was measured according to the manufacturer's instruction. Data presented as mean ± SD from triplicates. * p

Techniques Used: Activation Assay, Transfection

ROS accumulation due to mitochondrial defects accounts for PML-mediated Nrf2 regulations. (A) ROS accumulation in Pml +/+ and Pml −/− MEFs. Equal numbers of Pml +/+ and Pml −/− MEFs were cultured in a 96-well plate. The ROS levels were measured according to manufacturer's instruction. Data presented as mean ± SD from triplicates. ** p
Figure Legend Snippet: ROS accumulation due to mitochondrial defects accounts for PML-mediated Nrf2 regulations. (A) ROS accumulation in Pml +/+ and Pml −/− MEFs. Equal numbers of Pml +/+ and Pml −/− MEFs were cultured in a 96-well plate. The ROS levels were measured according to manufacturer's instruction. Data presented as mean ± SD from triplicates. ** p

Techniques Used: Cell Culture

PML negatively regulates Nrf2 protein abundance and its downstream target genes. (A) A heat map of significantly altered genes (greater than twofold, p
Figure Legend Snippet: PML negatively regulates Nrf2 protein abundance and its downstream target genes. (A) A heat map of significantly altered genes (greater than twofold, p

Techniques Used:

PML antagonizes transactivating activity of Nrf2. (A) qRT-PCR analysis of the mRNA levels of Nrf2 target genes and Pml in Pml +/+ and Pml −/− MEFs. Total RNA was prepared from Pml +/+ and Pml −/− MEFs and reverse transcribed into cDNA, which was used as a template for qRT-PCR analysis. Values normalized to the amount of each mRNA in Pml +/+ MEFs. Data presented as mean ± SD from triplicates. * p
Figure Legend Snippet: PML antagonizes transactivating activity of Nrf2. (A) qRT-PCR analysis of the mRNA levels of Nrf2 target genes and Pml in Pml +/+ and Pml −/− MEFs. Total RNA was prepared from Pml +/+ and Pml −/− MEFs and reverse transcribed into cDNA, which was used as a template for qRT-PCR analysis. Values normalized to the amount of each mRNA in Pml +/+ MEFs. Data presented as mean ± SD from triplicates. * p

Techniques Used: Activity Assay, Quantitative RT-PCR

PML inhibits nuclear accumulation of Nrf2. (A) Subcellular fractionation and immunoblotting analysis of Pml +/+ and Pml −/− MEFs. Nuclear and cytoplasmic fractions prepared from Pml +/+ and Pml −/− MEFs were subjected to immunoblotting analysis with the indicated antibodies. Lamin B and α-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively. Relative intensities of the bands are normalized to both loading control and Pml +/+ . N, nucleus; C, cytoplasm. (B) Immunofluorescence analysis of MEFs. Cells were immunostained with anti-PML and anti-Nrf2 antibodies, and images were taken by a fluorescence microscope. DAPI-stained nuclei (a, d); PML (b, e); Nrf2 (c, f). Scale bar, 20 μm. (C) Subcellular fractionation and immunoblotting analysis of HeLa cells with PML overexpression. HeLa cells were transfected with plasmids expressing HA-tagged PML4. Nuclear and cytoplasmic fractions prepared from transfected HeLa cells were subjected to immunoblotting analysis with the indicated antibodies. Relative intensities of the bands are normalized to both loading control and vector control. N, nucleus; C, cytoplasm. (D) Immunofluorescence analysis of HeLa cells with PML1 or PML4 overexpression. HeLa cells were transfected with plasmids expressing HA-tagged PML1 or PML4. Cells were immunostained with anti-HA and anti-Nrf2 antibodies, and images were taken on a fluorescence microscope. DAPI-stained nuclei (a, d, g); HA-tagged PML (b, e, h); endogenous Nrf2 (c, f, i). The arrows mark cells expressing transfected PML. Scale bar, 20 μm. (E) Immunofluorescence analysis of HUVECs with PML1 or PML4 overexpression. The experiments were performed as described in D. (F) The effects of nuclear and cytoplasmic mutants of PML4 overexpression on endogenous Nrf2 protein abundance in HeLa cells. HeLa cells were transfected with plasmids expressing HA-tagged PML4 (wild type), PML4 (K487R), and NLS-PML4 (K487R). Dividing line marks edges of different parts of the same gel.
Figure Legend Snippet: PML inhibits nuclear accumulation of Nrf2. (A) Subcellular fractionation and immunoblotting analysis of Pml +/+ and Pml −/− MEFs. Nuclear and cytoplasmic fractions prepared from Pml +/+ and Pml −/− MEFs were subjected to immunoblotting analysis with the indicated antibodies. Lamin B and α-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively. Relative intensities of the bands are normalized to both loading control and Pml +/+ . N, nucleus; C, cytoplasm. (B) Immunofluorescence analysis of MEFs. Cells were immunostained with anti-PML and anti-Nrf2 antibodies, and images were taken by a fluorescence microscope. DAPI-stained nuclei (a, d); PML (b, e); Nrf2 (c, f). Scale bar, 20 μm. (C) Subcellular fractionation and immunoblotting analysis of HeLa cells with PML overexpression. HeLa cells were transfected with plasmids expressing HA-tagged PML4. Nuclear and cytoplasmic fractions prepared from transfected HeLa cells were subjected to immunoblotting analysis with the indicated antibodies. Relative intensities of the bands are normalized to both loading control and vector control. N, nucleus; C, cytoplasm. (D) Immunofluorescence analysis of HeLa cells with PML1 or PML4 overexpression. HeLa cells were transfected with plasmids expressing HA-tagged PML1 or PML4. Cells were immunostained with anti-HA and anti-Nrf2 antibodies, and images were taken on a fluorescence microscope. DAPI-stained nuclei (a, d, g); HA-tagged PML (b, e, h); endogenous Nrf2 (c, f, i). The arrows mark cells expressing transfected PML. Scale bar, 20 μm. (E) Immunofluorescence analysis of HUVECs with PML1 or PML4 overexpression. The experiments were performed as described in D. (F) The effects of nuclear and cytoplasmic mutants of PML4 overexpression on endogenous Nrf2 protein abundance in HeLa cells. HeLa cells were transfected with plasmids expressing HA-tagged PML4 (wild type), PML4 (K487R), and NLS-PML4 (K487R). Dividing line marks edges of different parts of the same gel.

Techniques Used: Fractionation, Immunofluorescence, Fluorescence, Microscopy, Staining, Over Expression, Transfection, Expressing, Plasmid Preparation

20) Product Images from "Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis"

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

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2014.49

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

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

21) Product Images from "A mechanism for the suppression of homologous recombination in G1 cells"

Article Title: A mechanism for the suppression of homologous recombination in G1 cells

Journal: Nature

doi: 10.1038/nature16142

USP11 opposes the activity of CRL3-KEAP1 a, Normal IgG or PALB2 immunoprecipitation (IP) of extracts derived from CPT-treated 293T cells of the indicated genotypes transfected with GFP-USP11 constructs. EV, empty vector; WT, wild type; CS, C318S. b , Clonogenic survival assays of 293T cells of the indicated genotypes treated with olaparib (mean ± s.d., N≥ 3). c , Normal IgG or PALB2 IP of extracts derived from CPT-treated 293T cells of the indicated genotypes. d , Immunoblots of deubiquitylation reactions containing ubiquitylated HA-tagged PALB2 (1-103) and increasing concentrations of GST-USP11 or its C318S (CS) mutant. USP2 was used as a control. e , Cell cycle-synchronized U2OS cells were irradiated (20 Gy dose) and processed for immunoblotting. f , Immunoblots of extracts from irradiated U2OS cells transfected with the indicated siRNAs. g , Fluorescence micrographs of G1-synchronized and irradiated (20 Gy) 53BP1Δ U2OS cells transfected with the indicated siRNAs. The percentage of cells with more than 5 γ-H2AX-colocalizing BRCA2 foci is indicated (mean ± s.d., N =3).
Figure Legend Snippet: USP11 opposes the activity of CRL3-KEAP1 a, Normal IgG or PALB2 immunoprecipitation (IP) of extracts derived from CPT-treated 293T cells of the indicated genotypes transfected with GFP-USP11 constructs. EV, empty vector; WT, wild type; CS, C318S. b , Clonogenic survival assays of 293T cells of the indicated genotypes treated with olaparib (mean ± s.d., N≥ 3). c , Normal IgG or PALB2 IP of extracts derived from CPT-treated 293T cells of the indicated genotypes. d , Immunoblots of deubiquitylation reactions containing ubiquitylated HA-tagged PALB2 (1-103) and increasing concentrations of GST-USP11 or its C318S (CS) mutant. USP2 was used as a control. e , Cell cycle-synchronized U2OS cells were irradiated (20 Gy dose) and processed for immunoblotting. f , Immunoblots of extracts from irradiated U2OS cells transfected with the indicated siRNAs. g , Fluorescence micrographs of G1-synchronized and irradiated (20 Gy) 53BP1Δ U2OS cells transfected with the indicated siRNAs. The percentage of cells with more than 5 γ-H2AX-colocalizing BRCA2 foci is indicated (mean ± s.d., N =3).

Techniques Used: Activity Assay, Immunoprecipitation, Derivative Assay, Cycling Probe Technology, Transfection, Construct, Plasmid Preparation, Western Blot, Mutagenesis, Irradiation, Fluorescence

22) Product Images from "KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿"

Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.05036-11

Knockdown of KPNA6 inhibits nuclear import of Keap1. (A) NIH 3T3 cells were cotransfected with either scrambled control siRNA or KPNA6 siRNA, along with an expression vector for Keap1. At 48 h after transfection, cells were treated with 5 nM LMB for the
Figure Legend Snippet: Knockdown of KPNA6 inhibits nuclear import of Keap1. (A) NIH 3T3 cells were cotransfected with either scrambled control siRNA or KPNA6 siRNA, along with an expression vector for Keap1. At 48 h after transfection, cells were treated with 5 nM LMB for the

Techniques Used: Expressing, Plasmid Preparation, Transfection

Overexpression of KPNA6 attenuates the inducible Nrf2 signaling in response to oxidative stress. (A) KPNA6 decreases the inducible Nrf2 protein level and the expression of its downstream detoxification genes without altering Keap1 protein levels. HEK293T
Figure Legend Snippet: Overexpression of KPNA6 attenuates the inducible Nrf2 signaling in response to oxidative stress. (A) KPNA6 decreases the inducible Nrf2 protein level and the expression of its downstream detoxification genes without altering Keap1 protein levels. HEK293T

Techniques Used: Over Expression, Expressing

The C-terminal Kelch domain of Keap1 mediates its nuclear entry. (A) Schematic of conserved domains in human Keap1 protein. Keap1 contains an N-terminal BTB domain, a C-terminal Kelch domain, and a linker region in between the two domains. The nuclear
Figure Legend Snippet: The C-terminal Kelch domain of Keap1 mediates its nuclear entry. (A) Schematic of conserved domains in human Keap1 protein. Keap1 contains an N-terminal BTB domain, a C-terminal Kelch domain, and a linker region in between the two domains. The nuclear

Techniques Used:

Schematic model of Nrf2 regulation by Keap1. Keap1 is a key regulator of the Nrf2-signaling pathway and serves as a molecular switch to turn the Nrf2-mediated antioxidant response on and off. (1) Oxidative stress or chemopreventive compounds cause a conformational
Figure Legend Snippet: Schematic model of Nrf2 regulation by Keap1. Keap1 is a key regulator of the Nrf2-signaling pathway and serves as a molecular switch to turn the Nrf2-mediated antioxidant response on and off. (1) Oxidative stress or chemopreventive compounds cause a conformational

Techniques Used:

The nuclear import of Keap1 occurs at its physiological protein level and is not dependent on Nrf1 or Nrf2. (A) GFP-tagged Keap1 proteins were expressed at levels similar to the levels of endogenous Keap1 in the stable MEF cell lines. Keap1 −/−
Figure Legend Snippet: The nuclear import of Keap1 occurs at its physiological protein level and is not dependent on Nrf1 or Nrf2. (A) GFP-tagged Keap1 proteins were expressed at levels similar to the levels of endogenous Keap1 in the stable MEF cell lines. Keap1 −/−

Techniques Used:

KPNA6 interacts with the Kelch domain of Keap1. (A) KPNA6 binds Keap1 in vitro . Purified His-tagged Keap1 proteins were incubated with the indicated in vitro -translated (IVT) 35 S-labeled proteins of the importin family, followed by pulldown with Ni-NTA
Figure Legend Snippet: KPNA6 interacts with the Kelch domain of Keap1. (A) KPNA6 binds Keap1 in vitro . Purified His-tagged Keap1 proteins were incubated with the indicated in vitro -translated (IVT) 35 S-labeled proteins of the importin family, followed by pulldown with Ni-NTA

Techniques Used: In Vitro, Purification, Incubation, Labeling

Overexpression of KPNA6 facilitates nuclear import of Keap1. (A) NIH 3T3 cells were transfected with an expression vector for Keap1 with or without a vector for Myc-tagged KPNA6. Cells were treated with 5 nM LMB for the indicated times. After fixation
Figure Legend Snippet: Overexpression of KPNA6 facilitates nuclear import of Keap1. (A) NIH 3T3 cells were transfected with an expression vector for Keap1 with or without a vector for Myc-tagged KPNA6. Cells were treated with 5 nM LMB for the indicated times. After fixation

Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation

23) Product Images from "Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity"

Article Title: Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.150

PTP1B deficiency protects hepatocytes against GSH depletion and elevation of ROS; effect on nuclear Nrf2 accumulation. PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with various doses of APAP for different time-periods. ( a ) Analysis of GSH content (4 h), ROS levels (6 h) and GPx and GR activity (16 h) in three independent experiments. * P
Figure Legend Snippet: PTP1B deficiency protects hepatocytes against GSH depletion and elevation of ROS; effect on nuclear Nrf2 accumulation. PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with various doses of APAP for different time-periods. ( a ) Analysis of GSH content (4 h), ROS levels (6 h) and GPx and GR activity (16 h) in three independent experiments. * P

Techniques Used: Activity Assay

PTP1B modulates GSK3 β /Src-Fyn-mediated Nrf2 nuclear accumulation in APAP-treated hepatocytes. ( a ) PTP1B +/+ immortalized hepatocytes were transfected with control or GSK3 β small interfering RNAs (siRNAs) (25 nM) for 48 h, followed by stimulation with 1 mM APAP. Nuclear and cytosolic extracts were analyzed by western blot with antibodies against Nrf2, Fyn and Src. ( b , upper panel) PTP1B +/+ immortalized hepatocytes were treated with PP2 (5 μ M) for 30 min following 1 mM APAP for different periods. Nrf2 in nuclear extracts was analyzed by western blot. (Lower panel) SYF −/− MEFs were treated with 1 mM APAP for different periods and Nrf2 was analyzed in nuclear extracts by western blot. ( c ) PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with 1 mM APAP for different periods. Phosphorylated GSK3β (Tyr216) was analyzed in cytosolic extracts by western blot. Fyn and Src were analyzed in nuclear extracts. Similar results were obtained in three independent experiments
Figure Legend Snippet: PTP1B modulates GSK3 β /Src-Fyn-mediated Nrf2 nuclear accumulation in APAP-treated hepatocytes. ( a ) PTP1B +/+ immortalized hepatocytes were transfected with control or GSK3 β small interfering RNAs (siRNAs) (25 nM) for 48 h, followed by stimulation with 1 mM APAP. Nuclear and cytosolic extracts were analyzed by western blot with antibodies against Nrf2, Fyn and Src. ( b , upper panel) PTP1B +/+ immortalized hepatocytes were treated with PP2 (5 μ M) for 30 min following 1 mM APAP for different periods. Nrf2 in nuclear extracts was analyzed by western blot. (Lower panel) SYF −/− MEFs were treated with 1 mM APAP for different periods and Nrf2 was analyzed in nuclear extracts by western blot. ( c ) PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with 1 mM APAP for different periods. Phosphorylated GSK3β (Tyr216) was analyzed in cytosolic extracts by western blot. Fyn and Src were analyzed in nuclear extracts. Similar results were obtained in three independent experiments

Techniques Used: Transfection, Western Blot

Beneficial effects of PTP1B deficiency on the induction of Nrf2-mediated antioxidant response and survival signaling in the liver. PTP1B +/+ and PTP1B −/− mice were injected with 300 mg/kg APAP or saline for 3 or 6 h. ( a ) Western blot analysis of Nrf2 in nuclear extracts and HO-1, phospho (p)-JNK and JNK in total liver extracts. ( b ) GPx, HO-1, GCL-M, GCL-C and NQO1 mRNA levels determined by quantitative real-time polymerase chain reaction (qRT-PCR) at 3 and 6 h after APAP injection. * P
Figure Legend Snippet: Beneficial effects of PTP1B deficiency on the induction of Nrf2-mediated antioxidant response and survival signaling in the liver. PTP1B +/+ and PTP1B −/− mice were injected with 300 mg/kg APAP or saline for 3 or 6 h. ( a ) Western blot analysis of Nrf2 in nuclear extracts and HO-1, phospho (p)-JNK and JNK in total liver extracts. ( b ) GPx, HO-1, GCL-M, GCL-C and NQO1 mRNA levels determined by quantitative real-time polymerase chain reaction (qRT-PCR) at 3 and 6 h after APAP injection. * P

Techniques Used: Mouse Assay, Injection, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

24) Product Images from "Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis"

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

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2014.49

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

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

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

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

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

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

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

Techniques Used: Stable Transfection, Expressing, Trypan Blue Exclusion Assay

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

Techniques Used: Stable Transfection, Expressing, Trypan Blue Exclusion Assay

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

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

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

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

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

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

25) Product Images from "Disturbance of redox status enhances radiosensitivity of hepatocellular carcinoma"

Article Title: Disturbance of redox status enhances radiosensitivity of hepatocellular carcinoma

Journal: American Journal of Cancer Research

doi:

Effects of ISL on Keap-1 and Nrf-2 on HepG2 cells. A. After various times of ISL treatment, expressions of Keap-1 and Nrf-2 mRNA were measured by RT-PCR. β-Actin was used as a standard. B. After various time treatments with ISL, expressions of Keap-1 and Nrf-2 proteins were measured by western blot. C. After various time treatments with ISL, ubiquitination of Nrf2 was measured by immunoprecipitation. All data are expressed as mean ± SEM from three independent experiments. * p
Figure Legend Snippet: Effects of ISL on Keap-1 and Nrf-2 on HepG2 cells. A. After various times of ISL treatment, expressions of Keap-1 and Nrf-2 mRNA were measured by RT-PCR. β-Actin was used as a standard. B. After various time treatments with ISL, expressions of Keap-1 and Nrf-2 proteins were measured by western blot. C. After various time treatments with ISL, ubiquitination of Nrf2 was measured by immunoprecipitation. All data are expressed as mean ± SEM from three independent experiments. * p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation

26) Product Images from "Ethanol Impairs NRF2/Antioxidant and Growth Signaling in the Intact Placenta In Vivo and in Human Trophoblasts"

Article Title: Ethanol Impairs NRF2/Antioxidant and Growth Signaling in the Intact Placenta In Vivo and in Human Trophoblasts

Journal: Biomolecules

doi: 10.3390/biom9110669

Effect of intrauterine alcohol exposure on NRF2 and KEAP1 protein expression. ( A ) A representative immunoblot image detecting placental NRF2 and LAMIN B1 protein expression in nuclear fraction (upper panel) and its quantification (bottom panel) from air (C) and IEV (E) exposed rats (hours) (total n = 12; n = 4 each from 3 different litters); ( B ) immunodetection of placental NRF2 and GAPDH protein in the cytosol of air (C) and IEV (E) exposed pregnant rats measured by Western blotting (upper panel) and the corresponding quantification of NRF2 protein levels normalized to the reference protein, GAPDH (bottom panel) (total n = 12; n = 4 each from 3 different litters); ( C ) Western blot image of KEAP1 and LAMIN B1 protein in placenta obtained from air (C) or vapor based ethanol (IEV)-exposed pregnant dams (upper panel) and the image densities of KEAP1 relative to LAMIN B1 (bottom panel) (total n = 12; n = 4 each from 3 different litters); ( D ) representative immunoblots demonstrating the expression of KEAP1 protein, with GAPDH as a loading control in placenta of control and intrauterine alcohol-exposed pregnant rats (upper panel) and the quantification of immunoblots of KEAP1 normalized to GAPDH (bottom panel). Values represent the mean ± SEM. * p
Figure Legend Snippet: Effect of intrauterine alcohol exposure on NRF2 and KEAP1 protein expression. ( A ) A representative immunoblot image detecting placental NRF2 and LAMIN B1 protein expression in nuclear fraction (upper panel) and its quantification (bottom panel) from air (C) and IEV (E) exposed rats (hours) (total n = 12; n = 4 each from 3 different litters); ( B ) immunodetection of placental NRF2 and GAPDH protein in the cytosol of air (C) and IEV (E) exposed pregnant rats measured by Western blotting (upper panel) and the corresponding quantification of NRF2 protein levels normalized to the reference protein, GAPDH (bottom panel) (total n = 12; n = 4 each from 3 different litters); ( C ) Western blot image of KEAP1 and LAMIN B1 protein in placenta obtained from air (C) or vapor based ethanol (IEV)-exposed pregnant dams (upper panel) and the image densities of KEAP1 relative to LAMIN B1 (bottom panel) (total n = 12; n = 4 each from 3 different litters); ( D ) representative immunoblots demonstrating the expression of KEAP1 protein, with GAPDH as a loading control in placenta of control and intrauterine alcohol-exposed pregnant rats (upper panel) and the quantification of immunoblots of KEAP1 normalized to GAPDH (bottom panel). Values represent the mean ± SEM. * p

Techniques Used: Expressing, Immunodetection, Western Blot

27) Product Images from "Defective protein repair under methionine sulfoxide A deletion drives autophagy and ARE-dependent gene transcription"

Article Title: Defective protein repair under methionine sulfoxide A deletion drives autophagy and ARE-dependent gene transcription

Journal: Redox Biology

doi: 10.1016/j.redox.2018.04.001

Elevated Nrf2 protein expression under MsrA deficiency is due to protein stabilization rather than increased transcription. (A, B) Representative immunoblot (A) and quantification (B) for Nrf2 protein levels in whole cell lysates from MsrA-/- and WT VSMC; n = 3 biological replicates. (C) Nrf2 mRNA levels in VSMC by qRT-PCR; n = 5 biological replicates. (D) Representative immunoprecipitation of Nrf2 followed by immunoblot for ubiquitin in MsrA-/- and WT VSMC. IgG: IP with IgG, WT + MC132: IP with anti-Nrf2 in WT VSMC incubated with MG132, WCL: whole cell lysate of WT VSMC as controls. (E) Quantification of (D); n = 7 biological replicates. (F) p62 mRNA levels by qRT-PCR in aortic samples WT, MsrA-/- and MsrA-/- x Nrf2-/- mice; n = 7, 9 biological replicates. (G) Representative Immunoblots for Nrf2 and GAPDH in aortic samples from WT, MsrA-/- and MsrA-/- x Nrf2-/- mice. (H) Quantification of (G) n = 7 biological replicates. (E) * p
Figure Legend Snippet: Elevated Nrf2 protein expression under MsrA deficiency is due to protein stabilization rather than increased transcription. (A, B) Representative immunoblot (A) and quantification (B) for Nrf2 protein levels in whole cell lysates from MsrA-/- and WT VSMC; n = 3 biological replicates. (C) Nrf2 mRNA levels in VSMC by qRT-PCR; n = 5 biological replicates. (D) Representative immunoprecipitation of Nrf2 followed by immunoblot for ubiquitin in MsrA-/- and WT VSMC. IgG: IP with IgG, WT + MC132: IP with anti-Nrf2 in WT VSMC incubated with MG132, WCL: whole cell lysate of WT VSMC as controls. (E) Quantification of (D); n = 7 biological replicates. (F) p62 mRNA levels by qRT-PCR in aortic samples WT, MsrA-/- and MsrA-/- x Nrf2-/- mice; n = 7, 9 biological replicates. (G) Representative Immunoblots for Nrf2 and GAPDH in aortic samples from WT, MsrA-/- and MsrA-/- x Nrf2-/- mice. (H) Quantification of (G) n = 7 biological replicates. (E) * p

Techniques Used: Expressing, Quantitative RT-PCR, Immunoprecipitation, Incubation, Mouse Assay, Western Blot

28) Product Images from "Control of antioxidative response by the tumor suppressor protein PML through regulating Nrf2 activity"

Article Title: Control of antioxidative response by the tumor suppressor protein PML through regulating Nrf2 activity

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E13-11-0692

The effects of SFN on abundance and subcellular distribution of PML. (A) Immunoblotting analysis of HUVECs treated with different concentrations of SFN. HUVECs were treated with SFN for 1 h at 0, 10, 20, 40, or 80 μM final concentration. Cell extracts were analyzed by immunoblotting with the indicated antibodies. β-Actin was used as loading control. (B) Immunoblotting analysis of HUVECs treated with SFN. HUVECs were treated with vehicle control (DMSO) or SFN at 10 or 20 μM for 0.5, 1, 4, or 8 h. Relative intensities of the bands are normalized to both loading control and DMSO within each time point. (C) Subcellular fractionation and immunoblotting analysis of HUVECs treated with SFN. HUVECs were treated with vehicle control (DMSO) or SFN at 10 or 20 μM for 0.5, 1, 4, or 8 h. Nuclear and cytoplasmic fractions prepared were subjected to immunoblotting analysis with the indicated antibodies. Lamin B and α-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively. Relative intensities of the bands are normalized to both loading control and DMSO within each time point. (D) Immunofluorescence analysis of HUVECs treated with SFN. HUVECs were treated with vehicle control (DMSO) or SFN at 10 μM for 1 h. Cells were immunostained with anti-PML antibodies, and images were taken on a fluorescence microscope. Nuclei were stained with DAPI, and PML NBs are shown in green. Statistical analysis of the PML NB number and nuclear fluorescence intensity. n > 150 per group. * p
Figure Legend Snippet: The effects of SFN on abundance and subcellular distribution of PML. (A) Immunoblotting analysis of HUVECs treated with different concentrations of SFN. HUVECs were treated with SFN for 1 h at 0, 10, 20, 40, or 80 μM final concentration. Cell extracts were analyzed by immunoblotting with the indicated antibodies. β-Actin was used as loading control. (B) Immunoblotting analysis of HUVECs treated with SFN. HUVECs were treated with vehicle control (DMSO) or SFN at 10 or 20 μM for 0.5, 1, 4, or 8 h. Relative intensities of the bands are normalized to both loading control and DMSO within each time point. (C) Subcellular fractionation and immunoblotting analysis of HUVECs treated with SFN. HUVECs were treated with vehicle control (DMSO) or SFN at 10 or 20 μM for 0.5, 1, 4, or 8 h. Nuclear and cytoplasmic fractions prepared were subjected to immunoblotting analysis with the indicated antibodies. Lamin B and α-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively. Relative intensities of the bands are normalized to both loading control and DMSO within each time point. (D) Immunofluorescence analysis of HUVECs treated with SFN. HUVECs were treated with vehicle control (DMSO) or SFN at 10 μM for 1 h. Cells were immunostained with anti-PML antibodies, and images were taken on a fluorescence microscope. Nuclei were stained with DAPI, and PML NBs are shown in green. Statistical analysis of the PML NB number and nuclear fluorescence intensity. n > 150 per group. * p

Techniques Used: Concentration Assay, Fractionation, Immunofluorescence, Fluorescence, Microscopy, Staining

PML inhibits nuclear accumulation of Nrf2. (A) Subcellular fractionation and immunoblotting analysis of Pml +/+ and Pml −/− MEFs. Nuclear and cytoplasmic fractions prepared from Pml +/+ and Pml −/− MEFs were subjected to immunoblotting analysis with the indicated antibodies. Lamin B and α-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively. Relative intensities of the bands are normalized to both loading control and Pml +/+ . N, nucleus; C, cytoplasm. (B) Immunofluorescence analysis of MEFs. Cells were immunostained with anti-PML and anti-Nrf2 antibodies, and images were taken by a fluorescence microscope. DAPI-stained nuclei (a, d); PML (b, e); Nrf2 (c, f). Scale bar, 20 μm. (C) Subcellular fractionation and immunoblotting analysis of HeLa cells with PML overexpression. HeLa cells were transfected with plasmids expressing HA-tagged PML4. Nuclear and cytoplasmic fractions prepared from transfected HeLa cells were subjected to immunoblotting analysis with the indicated antibodies. Relative intensities of the bands are normalized to both loading control and vector control. N, nucleus; C, cytoplasm. (D) Immunofluorescence analysis of HeLa cells with PML1 or PML4 overexpression. HeLa cells were transfected with plasmids expressing HA-tagged PML1 or PML4. Cells were immunostained with anti-HA and anti-Nrf2 antibodies, and images were taken on a fluorescence microscope. DAPI-stained nuclei (a, d, g); HA-tagged PML (b, e, h); endogenous Nrf2 (c, f, i). The arrows mark cells expressing transfected PML. Scale bar, 20 μm. (E) Immunofluorescence analysis of HUVECs with PML1 or PML4 overexpression. The experiments were performed as described in D. (F) The effects of nuclear and cytoplasmic mutants of PML4 overexpression on endogenous Nrf2 protein abundance in HeLa cells. HeLa cells were transfected with plasmids expressing HA-tagged PML4 (wild type), PML4 (K487R), and NLS-PML4 (K487R). Dividing line marks edges of different parts of the same gel.
Figure Legend Snippet: PML inhibits nuclear accumulation of Nrf2. (A) Subcellular fractionation and immunoblotting analysis of Pml +/+ and Pml −/− MEFs. Nuclear and cytoplasmic fractions prepared from Pml +/+ and Pml −/− MEFs were subjected to immunoblotting analysis with the indicated antibodies. Lamin B and α-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively. Relative intensities of the bands are normalized to both loading control and Pml +/+ . N, nucleus; C, cytoplasm. (B) Immunofluorescence analysis of MEFs. Cells were immunostained with anti-PML and anti-Nrf2 antibodies, and images were taken by a fluorescence microscope. DAPI-stained nuclei (a, d); PML (b, e); Nrf2 (c, f). Scale bar, 20 μm. (C) Subcellular fractionation and immunoblotting analysis of HeLa cells with PML overexpression. HeLa cells were transfected with plasmids expressing HA-tagged PML4. Nuclear and cytoplasmic fractions prepared from transfected HeLa cells were subjected to immunoblotting analysis with the indicated antibodies. Relative intensities of the bands are normalized to both loading control and vector control. N, nucleus; C, cytoplasm. (D) Immunofluorescence analysis of HeLa cells with PML1 or PML4 overexpression. HeLa cells were transfected with plasmids expressing HA-tagged PML1 or PML4. Cells were immunostained with anti-HA and anti-Nrf2 antibodies, and images were taken on a fluorescence microscope. DAPI-stained nuclei (a, d, g); HA-tagged PML (b, e, h); endogenous Nrf2 (c, f, i). The arrows mark cells expressing transfected PML. Scale bar, 20 μm. (E) Immunofluorescence analysis of HUVECs with PML1 or PML4 overexpression. The experiments were performed as described in D. (F) The effects of nuclear and cytoplasmic mutants of PML4 overexpression on endogenous Nrf2 protein abundance in HeLa cells. HeLa cells were transfected with plasmids expressing HA-tagged PML4 (wild type), PML4 (K487R), and NLS-PML4 (K487R). Dividing line marks edges of different parts of the same gel.

Techniques Used: Fractionation, Immunofluorescence, Fluorescence, Microscopy, Staining, Over Expression, Transfection, Expressing, Plasmid Preparation

29) Product Images from "Hydroxylated Dimeric Naphthoquinones Increase the Generation of Reactive Oxygen Species, Induce Apoptosis of Acute Myeloid Leukemia Cells and Are Not Substrates of the Multidrug Resistance Proteins ABCB1 and ABCG2"

Article Title: Hydroxylated Dimeric Naphthoquinones Increase the Generation of Reactive Oxygen Species, Induce Apoptosis of Acute Myeloid Leukemia Cells and Are Not Substrates of the Multidrug Resistance Proteins ABCB1 and ABCG2

Journal: Pharmaceuticals

doi: 10.3390/ph9010004

BiQ-1 induced apoptosis of AML cells as measured by Western Blot. ( A ) BiQ-1 treatment of MOLM-14 cells resulted in reduced Mcl-1 expression at 6 and 24 h post-treatment; ( B ) In contrast, at 6 and 24 h post-treatment in THP-1, a concentration-dependent increase in Mcl-1 expression was observed. ( A , B ) In MOLM-14 and THP-1, caspase-3 cleavage was observed after 6 h of treatment with 20 µM and 40 µM BiQ-1, respectively. Caspase-3 cleavage was maintained at 24 h in MOLM-14, but not THP-1; ( C – E ) In concentrations ranging from 10 µM to 20 µM, BiQ-1 induced caspase-3 cleavage within 6 h in primary AML cells from patients.
Figure Legend Snippet: BiQ-1 induced apoptosis of AML cells as measured by Western Blot. ( A ) BiQ-1 treatment of MOLM-14 cells resulted in reduced Mcl-1 expression at 6 and 24 h post-treatment; ( B ) In contrast, at 6 and 24 h post-treatment in THP-1, a concentration-dependent increase in Mcl-1 expression was observed. ( A , B ) In MOLM-14 and THP-1, caspase-3 cleavage was observed after 6 h of treatment with 20 µM and 40 µM BiQ-1, respectively. Caspase-3 cleavage was maintained at 24 h in MOLM-14, but not THP-1; ( C – E ) In concentrations ranging from 10 µM to 20 µM, BiQ-1 induced caspase-3 cleavage within 6 h in primary AML cells from patients.

Techniques Used: Western Blot, Expressing, Concentration Assay

30) Product Images from "Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity"

Article Title: Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.150

PTP1B deficiency protects hepatocytes against GSH depletion and elevation of ROS; effect on nuclear Nrf2 accumulation. PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with various doses of APAP for different time-periods. ( a ) Analysis of GSH content (4 h), ROS levels (6 h) and GPx and GR activity (16 h) in three independent experiments. * P
Figure Legend Snippet: PTP1B deficiency protects hepatocytes against GSH depletion and elevation of ROS; effect on nuclear Nrf2 accumulation. PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with various doses of APAP for different time-periods. ( a ) Analysis of GSH content (4 h), ROS levels (6 h) and GPx and GR activity (16 h) in three independent experiments. * P

Techniques Used: Activity Assay

PTP1B modulates GSK3 β /Src-Fyn-mediated Nrf2 nuclear accumulation in APAP-treated hepatocytes. ( a ) PTP1B +/+ immortalized hepatocytes were transfected with control or GSK3 β small interfering RNAs (siRNAs) (25 nM) for 48 h, followed by stimulation with 1 mM APAP. Nuclear and cytosolic extracts were analyzed by western blot with antibodies against Nrf2, Fyn and Src. ( b , upper panel) PTP1B +/+ immortalized hepatocytes were treated with PP2 (5 μ M) for 30 min following 1 mM APAP for different periods. Nrf2 in nuclear extracts was analyzed by western blot. (Lower panel) SYF −/− MEFs were treated with 1 mM APAP for different periods and Nrf2 was analyzed in nuclear extracts by western blot. ( c ) PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with 1 mM APAP for different periods. Phosphorylated GSK3β (Tyr216) was analyzed in cytosolic extracts by western blot. Fyn and Src were analyzed in nuclear extracts. Similar results were obtained in three independent experiments
Figure Legend Snippet: PTP1B modulates GSK3 β /Src-Fyn-mediated Nrf2 nuclear accumulation in APAP-treated hepatocytes. ( a ) PTP1B +/+ immortalized hepatocytes were transfected with control or GSK3 β small interfering RNAs (siRNAs) (25 nM) for 48 h, followed by stimulation with 1 mM APAP. Nuclear and cytosolic extracts were analyzed by western blot with antibodies against Nrf2, Fyn and Src. ( b , upper panel) PTP1B +/+ immortalized hepatocytes were treated with PP2 (5 μ M) for 30 min following 1 mM APAP for different periods. Nrf2 in nuclear extracts was analyzed by western blot. (Lower panel) SYF −/− MEFs were treated with 1 mM APAP for different periods and Nrf2 was analyzed in nuclear extracts by western blot. ( c ) PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with 1 mM APAP for different periods. Phosphorylated GSK3β (Tyr216) was analyzed in cytosolic extracts by western blot. Fyn and Src were analyzed in nuclear extracts. Similar results were obtained in three independent experiments

Techniques Used: Transfection, Western Blot

PTP1B-deficient primary hepatocytes are protected against APAP-induced cell death. ( a , left panel) Wild-type (PTP1B +/+ ) mouse primary hepatocytes were treated with 10 mM APAP for various time-periods. The expression of PTP1B was analyzed by western blot. * P
Figure Legend Snippet: PTP1B-deficient primary hepatocytes are protected against APAP-induced cell death. ( a , left panel) Wild-type (PTP1B +/+ ) mouse primary hepatocytes were treated with 10 mM APAP for various time-periods. The expression of PTP1B was analyzed by western blot. * P

Techniques Used: Expressing, Western Blot

Beneficial effects of PTP1B deficiency on the induction of Nrf2-mediated antioxidant response and survival signaling in the liver. PTP1B +/+ and PTP1B −/− mice were injected with 300 mg/kg APAP or saline for 3 or 6 h. ( a ) Western blot analysis of Nrf2 in nuclear extracts and HO-1, phospho (p)-JNK and JNK in total liver extracts. ( b ) GPx, HO-1, GCL-M, GCL-C and NQO1 mRNA levels determined by quantitative real-time polymerase chain reaction (qRT-PCR) at 3 and 6 h after APAP injection. * P
Figure Legend Snippet: Beneficial effects of PTP1B deficiency on the induction of Nrf2-mediated antioxidant response and survival signaling in the liver. PTP1B +/+ and PTP1B −/− mice were injected with 300 mg/kg APAP or saline for 3 or 6 h. ( a ) Western blot analysis of Nrf2 in nuclear extracts and HO-1, phospho (p)-JNK and JNK in total liver extracts. ( b ) GPx, HO-1, GCL-M, GCL-C and NQO1 mRNA levels determined by quantitative real-time polymerase chain reaction (qRT-PCR) at 3 and 6 h after APAP injection. * P

Techniques Used: Mouse Assay, Injection, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Effect of PTP1B deficiency in stress and survival signaling in hepatocytes. ( a , left panel) PTP1B +/+ and PTP1B −/− mouse primary hepatocytes were treated with APAP (10 mM) for various time periods. Total cell lysates were analyzed by western blot with the antibodies against phospho (p)-JNK1/2, JNK1/2, phospho-p38 MAPK and p38 MAPK. Representative autoradiograms corresponding to three independent experiments are shown. (Right panel) PTP1B +/+ and PTP1B −/− mouse primary hepatocytes were treated with APAP (5 and 10 mM) for 16 h. Total cell lysates were analyzed by western blot with the antibodies against phospho (p)-IGFIR, IGFIR, IRS1, IRS2, phospho-Akt, Akt, BclxL, Mcl1 and β -actin as a loading control. Representative autoradiograms corresponding to three independent experiments are shown. ( b ) PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with various doses of APAP for 16 h. Total cell lysates were analyzed by western blot with the indicated antibodies. Representative autoradiograms corresponding to three independent experiments are shown. ( c ) Immortalized PTP1B +/+ hepatocytes transfected with 10 nM of control or PTP1B small interfering RNA (siRNA) for 48 h were further treated with various doses of APAP for 16 h. Total cell lysates were analyzed by western blot with the indicated antibodies. Representative autoradiograms corresponding to three independent experiments are shown
Figure Legend Snippet: Effect of PTP1B deficiency in stress and survival signaling in hepatocytes. ( a , left panel) PTP1B +/+ and PTP1B −/− mouse primary hepatocytes were treated with APAP (10 mM) for various time periods. Total cell lysates were analyzed by western blot with the antibodies against phospho (p)-JNK1/2, JNK1/2, phospho-p38 MAPK and p38 MAPK. Representative autoradiograms corresponding to three independent experiments are shown. (Right panel) PTP1B +/+ and PTP1B −/− mouse primary hepatocytes were treated with APAP (5 and 10 mM) for 16 h. Total cell lysates were analyzed by western blot with the antibodies against phospho (p)-IGFIR, IGFIR, IRS1, IRS2, phospho-Akt, Akt, BclxL, Mcl1 and β -actin as a loading control. Representative autoradiograms corresponding to three independent experiments are shown. ( b ) PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with various doses of APAP for 16 h. Total cell lysates were analyzed by western blot with the indicated antibodies. Representative autoradiograms corresponding to three independent experiments are shown. ( c ) Immortalized PTP1B +/+ hepatocytes transfected with 10 nM of control or PTP1B small interfering RNA (siRNA) for 48 h were further treated with various doses of APAP for 16 h. Total cell lysates were analyzed by western blot with the indicated antibodies. Representative autoradiograms corresponding to three independent experiments are shown

Techniques Used: Western Blot, Transfection, Small Interfering RNA

PTP1B expression is increased during APAP-induced liver injury and in human primary hepatocytes treated with APAP. ( a ) Representative anti-PTP1B immunostaining of liver biopsy sections from patients with histologically normal liver (NL) or with an APAP overdose (APAP) ( n =7). Bar=50 μ m. ( b ) Human primary hepatocytes were treated with various doses of APAP (5–20 mM) for 24 h. Total cell lysates were analyzed by western blot with the antibodies against PTP1B and β -actin as a loading control. Autoradiograms corresponding were quantitated by scanning densitometry. Results are means±S.E.M. * P
Figure Legend Snippet: PTP1B expression is increased during APAP-induced liver injury and in human primary hepatocytes treated with APAP. ( a ) Representative anti-PTP1B immunostaining of liver biopsy sections from patients with histologically normal liver (NL) or with an APAP overdose (APAP) ( n =7). Bar=50 μ m. ( b ) Human primary hepatocytes were treated with various doses of APAP (5–20 mM) for 24 h. Total cell lysates were analyzed by western blot with the antibodies against PTP1B and β -actin as a loading control. Autoradiograms corresponding were quantitated by scanning densitometry. Results are means±S.E.M. * P

Techniques Used: Expressing, Immunostaining, Western Blot

PTP1B-deficient mice are protected against APAP-induced, inflammation oxidative stress and liver damage. PTP1B +/+ and PTP1B −/− mice were injected with 300 mg/kg APAP or saline for 6 or 24 h. ( a ) Survival curves after 24 h of APAP injection. ( b ) ALT activity at 6 and 24 h. ( c ) Representative images of whole livers from PTP1B +/+ and PTP1B −/− mice 24 h after APAP injection. ( d ) IL6, IL1 β and TNF α mRNA levels were determined by real-time polymerase chain reaction in livers from PTP1B +/+ and PTP1B −/− mice at 6 h after APAP injection. ( e ) Representative anti-PTP1B immunostaining (left panel) and hematoxylin and eosin staining (right panel) in livers from PTP1B +/+ and PTP1B −/− mice 6 h after APAP injection. Bar=100 μ m. ( f ) GSH (left panel), APAP–protein adducts (middle panel) and carbonylated protein levels (right panel) were analyzed in livers from PTP1B +/+ and PTP1B −/− mice at various time-periods after APAP injection. * P
Figure Legend Snippet: PTP1B-deficient mice are protected against APAP-induced, inflammation oxidative stress and liver damage. PTP1B +/+ and PTP1B −/− mice were injected with 300 mg/kg APAP or saline for 6 or 24 h. ( a ) Survival curves after 24 h of APAP injection. ( b ) ALT activity at 6 and 24 h. ( c ) Representative images of whole livers from PTP1B +/+ and PTP1B −/− mice 24 h after APAP injection. ( d ) IL6, IL1 β and TNF α mRNA levels were determined by real-time polymerase chain reaction in livers from PTP1B +/+ and PTP1B −/− mice at 6 h after APAP injection. ( e ) Representative anti-PTP1B immunostaining (left panel) and hematoxylin and eosin staining (right panel) in livers from PTP1B +/+ and PTP1B −/− mice 6 h after APAP injection. Bar=100 μ m. ( f ) GSH (left panel), APAP–protein adducts (middle panel) and carbonylated protein levels (right panel) were analyzed in livers from PTP1B +/+ and PTP1B −/− mice at various time-periods after APAP injection. * P

Techniques Used: Mouse Assay, Injection, Activity Assay, Real-time Polymerase Chain Reaction, Immunostaining, Staining

31) Product Images from "Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity"

Article Title: Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.150

Effect of PTP1B deficiency in stress and survival signaling in hepatocytes. ( a , left panel) PTP1B +/+ and PTP1B −/− mouse primary hepatocytes were treated with APAP (10 mM) for various time periods. Total cell lysates were analyzed by western blot with the antibodies against phospho (p)-JNK1/2, JNK1/2, phospho-p38 MAPK and p38 MAPK. Representative autoradiograms corresponding to three independent experiments are shown. (Right panel) PTP1B +/+ and PTP1B −/− mouse primary hepatocytes were treated with APAP (5 and 10 mM) for 16 h. Total cell lysates were analyzed by western blot with the antibodies against phospho (p)-IGFIR, IGFIR, IRS1, IRS2, phospho-Akt, Akt, BclxL, Mcl1 and β -actin as a loading control. Representative autoradiograms corresponding to three independent experiments are shown. ( b ) PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with various doses of APAP for 16 h. Total cell lysates were analyzed by western blot with the indicated antibodies. Representative autoradiograms corresponding to three independent experiments are shown. ( c ) Immortalized PTP1B +/+ hepatocytes transfected with 10 nM of control or PTP1B small interfering RNA (siRNA) for 48 h were further treated with various doses of APAP for 16 h. Total cell lysates were analyzed by western blot with the indicated antibodies. Representative autoradiograms corresponding to three independent experiments are shown
Figure Legend Snippet: Effect of PTP1B deficiency in stress and survival signaling in hepatocytes. ( a , left panel) PTP1B +/+ and PTP1B −/− mouse primary hepatocytes were treated with APAP (10 mM) for various time periods. Total cell lysates were analyzed by western blot with the antibodies against phospho (p)-JNK1/2, JNK1/2, phospho-p38 MAPK and p38 MAPK. Representative autoradiograms corresponding to three independent experiments are shown. (Right panel) PTP1B +/+ and PTP1B −/− mouse primary hepatocytes were treated with APAP (5 and 10 mM) for 16 h. Total cell lysates were analyzed by western blot with the antibodies against phospho (p)-IGFIR, IGFIR, IRS1, IRS2, phospho-Akt, Akt, BclxL, Mcl1 and β -actin as a loading control. Representative autoradiograms corresponding to three independent experiments are shown. ( b ) PTP1B +/+ and PTP1B −/− immortalized hepatocytes were treated with various doses of APAP for 16 h. Total cell lysates were analyzed by western blot with the indicated antibodies. Representative autoradiograms corresponding to three independent experiments are shown. ( c ) Immortalized PTP1B +/+ hepatocytes transfected with 10 nM of control or PTP1B small interfering RNA (siRNA) for 48 h were further treated with various doses of APAP for 16 h. Total cell lysates were analyzed by western blot with the indicated antibodies. Representative autoradiograms corresponding to three independent experiments are shown

Techniques Used: Western Blot, Transfection, Small Interfering RNA

32) Product Images from "A potent small-molecule inhibitor of the DCN1-UBC12 interaction that selectively blocks cullin 3 neddylation"

Article Title: A potent small-molecule inhibitor of the DCN1-UBC12 interaction that selectively blocks cullin 3 neddylation

Journal: Nature Communications

doi: 10.1038/s41467-017-01243-7

DI-591 selectively and rapidly inhibits neddylation of cullin 3. a Western blotting of neddylated and un-neddylated cullin family members and several representative well-known substrates of cullin CRLs in KYSE70 esophageal cancer cells and THLE2 immortalized liver cells. Cells were treated with DCN1 inhibitor DI-591 or NAE E1 inhibitor MLN4924 at indicated concentrations, or DI-591DD at 10 µM for 24 h. Protein levels of neddylated and un-neddylated cullin family members and several known substrates of cullin CRLs were examined by western blotting analysis. GAPDH was used as a loading control. b Inhibition kinetics of neddylation of cullin 1 and 3 by DI-591 and MLN4924 in THLE2 liver cells. Cells were treated with DI-591 at 10 µM or MLN4924 at 0.3 µM at indicated time points. Protein levels of neddylated and un-neddylated cullin 1 and 3 were examined by western blotting analysis. GAPDH was used as a loading control. c qRT-PCR analysis of mRNA levels of NRF2 and NRF2-reguated genes in THLE2 cells. Cells were treated with DI-591 at 10 µM, DI-591DD at 10 µM or DMSO for indicated time points. The relative mRNA levels of NRF2, NQO1 and HO-1 were examined by quantitative real-time RT-PCR assay. GAPDH was used as an internal control. The averages and standard deviations for each column were calculated from total nine samples obtained from three independent experiments (each experiment with triplicates). P -value from t-test: * P
Figure Legend Snippet: DI-591 selectively and rapidly inhibits neddylation of cullin 3. a Western blotting of neddylated and un-neddylated cullin family members and several representative well-known substrates of cullin CRLs in KYSE70 esophageal cancer cells and THLE2 immortalized liver cells. Cells were treated with DCN1 inhibitor DI-591 or NAE E1 inhibitor MLN4924 at indicated concentrations, or DI-591DD at 10 µM for 24 h. Protein levels of neddylated and un-neddylated cullin family members and several known substrates of cullin CRLs were examined by western blotting analysis. GAPDH was used as a loading control. b Inhibition kinetics of neddylation of cullin 1 and 3 by DI-591 and MLN4924 in THLE2 liver cells. Cells were treated with DI-591 at 10 µM or MLN4924 at 0.3 µM at indicated time points. Protein levels of neddylated and un-neddylated cullin 1 and 3 were examined by western blotting analysis. GAPDH was used as a loading control. c qRT-PCR analysis of mRNA levels of NRF2 and NRF2-reguated genes in THLE2 cells. Cells were treated with DI-591 at 10 µM, DI-591DD at 10 µM or DMSO for indicated time points. The relative mRNA levels of NRF2, NQO1 and HO-1 were examined by quantitative real-time RT-PCR assay. GAPDH was used as an internal control. The averages and standard deviations for each column were calculated from total nine samples obtained from three independent experiments (each experiment with triplicates). P -value from t-test: * P

Techniques Used: Western Blot, Inhibition, Quantitative RT-PCR

Cellular engagement of DCN proteins by DI-591. a Chemical structure of biotinylated compound 47 and its binding affinities to DCN1-5 proteins. b Pull-down of DCN1 and DCN2 protein by compound 47 and competition by DI-591 in KYSE70 cell lysates. Protein levels of DCN1, DCN2, cullin 1 and cullin 3 pulled down from KYSE70 cell lysates with 47 alone or in combination with DI-591 or DI-591DD were examined by western blotting analysis and specific antibodies. c Enhancement of thermal stability of DCN1 protein by DI-591 but not by DI-591DD in KYSE70 cells. Protein levels of DCN1 in KYSE70 cells treated with DI-591 at 10 µM, DI-591DD at 10 µM or DMSO for 1 h, followed by heating at different temperatures for 3 min were examined by western blotting analysis. GAPDH was used as a loading control. d Enhancement of thermal stability of DCN1 and DCN2 proteins by DI-591 but not by DI-591DD treatment in KYSE70 cells. Protein levels of DCN1 and DCN2 in KYSE70 cells treated with DI-591 and DI-591DD at the indicated concentrations for 1 h and then heated at 53 °C (DCN1) and 45 °C (DCN2) for 3 min were analyzed by western blot. GAPDH was used as a loading control. e Enhancement of thermal stability of DCN2 by DI-591 but not DI-591DD in KYSE70 cells. Protein levels of DCN2 in KYSE70 cells treated with DI-591 at 10 µM or DI-591DD at 10 µM or DMSO for 1 h, followed by heating at different temperature for 3 min were examined by western blotting analysis. GAPDH was used as a loading control. f Blockage of the association of DCN1 and UBC12 in cells by DI-591 but not by DI-591DD. KYSE70 cells were treated with DI-591 at 10 µM or DI-591DD at 10 µM or DMSO for 1 h. The basal protein levels of DCN1 and UBC12 in the cell lysates were examined by western blotting analysis. Protein levels of DCN1 and UBC12 in the cell lysates pulled down by an UBC12 antibody were examined by western blotting analysis
Figure Legend Snippet: Cellular engagement of DCN proteins by DI-591. a Chemical structure of biotinylated compound 47 and its binding affinities to DCN1-5 proteins. b Pull-down of DCN1 and DCN2 protein by compound 47 and competition by DI-591 in KYSE70 cell lysates. Protein levels of DCN1, DCN2, cullin 1 and cullin 3 pulled down from KYSE70 cell lysates with 47 alone or in combination with DI-591 or DI-591DD were examined by western blotting analysis and specific antibodies. c Enhancement of thermal stability of DCN1 protein by DI-591 but not by DI-591DD in KYSE70 cells. Protein levels of DCN1 in KYSE70 cells treated with DI-591 at 10 µM, DI-591DD at 10 µM or DMSO for 1 h, followed by heating at different temperatures for 3 min were examined by western blotting analysis. GAPDH was used as a loading control. d Enhancement of thermal stability of DCN1 and DCN2 proteins by DI-591 but not by DI-591DD treatment in KYSE70 cells. Protein levels of DCN1 and DCN2 in KYSE70 cells treated with DI-591 and DI-591DD at the indicated concentrations for 1 h and then heated at 53 °C (DCN1) and 45 °C (DCN2) for 3 min were analyzed by western blot. GAPDH was used as a loading control. e Enhancement of thermal stability of DCN2 by DI-591 but not DI-591DD in KYSE70 cells. Protein levels of DCN2 in KYSE70 cells treated with DI-591 at 10 µM or DI-591DD at 10 µM or DMSO for 1 h, followed by heating at different temperature for 3 min were examined by western blotting analysis. GAPDH was used as a loading control. f Blockage of the association of DCN1 and UBC12 in cells by DI-591 but not by DI-591DD. KYSE70 cells were treated with DI-591 at 10 µM or DI-591DD at 10 µM or DMSO for 1 h. The basal protein levels of DCN1 and UBC12 in the cell lysates were examined by western blotting analysis. Protein levels of DCN1 and UBC12 in the cell lysates pulled down by an UBC12 antibody were examined by western blotting analysis

Techniques Used: Binding Assay, Western Blot

Related Articles

Transduction:

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells
Article Snippet: .. Moreover, the number of cells in Nrf2 overexpressing middle-aged grafts was also higher (although not significantly, p > 0.05) compared with control middle-aged grafts transduced with just eGFP ( ). ..

Acetylene Reduction Assay:

Article Title: Digoxin sensitizes gemcitabine-resistant pancreatic cancer cells to gemcitabine via inhibiting Nrf2 signaling pathway
Article Snippet: Digoxin (purity > 97%), etoposide (purity > 99%), paclitaxel (purity > 99%), cisplatin (purity > 99%), 5-Fluorouracil (5-FU, purity > 99%), cytarabine (ara-C, purity > 99%), doxorubicin (purity > 99%) and MG132 (purity > 97%) were purchased from Selleck Chemicals (Houston, USA). .. Anti-NQO1, anti-HO-1 and anti-GCLC antibodies were obtained from Santa Cruz Biotechnology (Texas, USA).

Transfection:

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells
Article Snippet: .. Under these conditions, interestingly the survival of the cells ( ) was not significantly affected however, the proliferation substantially improved ( , p < 0.001, untreated versus Nrf2 transfected). .. We additionally also assessed DG NSPCs from newborn (postnatal day 0) Nrf2 knockout (Nrf2-/-) and WT (Nrf2+/+) mice.

MTT Assay:

Article Title: Digoxin sensitizes gemcitabine-resistant pancreatic cancer cells to gemcitabine via inhibiting Nrf2 signaling pathway
Article Snippet: 2.1 Materials Gemcitabine (purity > 98%), cycloheximide (purity > 93%) and MTT (purity > 98%) were purchased from Sigma-Aldrich (St. Louis, USA). .. Anti-NQO1, anti-HO-1 and anti-GCLC antibodies were obtained from Santa Cruz Biotechnology (Texas, USA).

Mouse Assay:

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells
Article Snippet: .. Here, a significant reduction in MCM2 , Sox2 , and GFAP/nestin ( ) expressing NSPCs was noted in the DG of the Nrf2-/- mice. .. Moreover, the number of Dcx+ newborn neurons was also significantly reduced in the Nrf2-/- mice compared with WT controls ( ).

Article Title: Loss of DJ-1 elicits retinal abnormalities, visual dysfunction, and increased oxidative stress in mice
Article Snippet: .. Immunoblots of retina/RPE lysates from 6 month-old mice revealed that DJ-1 KO l retinas displayed significant decreased immunoreactivity of red/green cone opsin, TH, Nrf2, ezrin and DJ-1 when compared to control lysates and normalized to the levels of GAPDH ( ). .. Quantification of these lysates ( ) demonstrated a 44% reduction in red/green opsin, a 40% reduction in TH, a 56% reduction in Nrf2 and a 83% reduction in ezrin signal intensity when comparing immunoreactivity in the control lysates.

other:

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells
Article Snippet: Finally, in the pattern separation test, the Nrf2-/- animals exhibited a compromised behavior as indicated by their substantially reduced exploration of the object in the novel context (p < 0.05, ) when compared with their WT counterparts.

Article Title: Antioxidant Properties of Fullerene Derivatives Depend on Their Chemical Structure: A Study of Two Fullerene Derivatives on HELFs
Article Snippet: NRF2 (erythroid-derived factor 2) is one of the main transcription factors that determine antioxidant response of the cells to the action of the internal and external ROS.

Article Title: ERBB2-modulated ATG4B and autophagic cell death in human ARPE19 during oxidative stress
Article Snippet: Accordingly, we evaluated NRF2 and autophagy involvement in ARPE-19 cells during oxidative stress.

Article Title: Nrf2-mediated anti-oxidant effects contribute to suppression of non-alcoholic steatohepatitis-associated hepatocellular carcinoma in murine model
Article Snippet: The results of this study support that Nrf2 and its related metabolites have protective effects on liver injury, inflammation, and tumorigenesis. ( , )

Article Title: Antioxidant Properties of Fullerene Derivatives Depend on Their Chemical Structure: A Study of Two Fullerene Derivatives on HELFs
Article Snippet: After 3 h with 4 nM of fullerene, the amount of NRF2 is reduced.

Article Title: Differential Regulation of the Three Eukaryotic mRNA Translation Initiation Factor (eIF) 4Gs by the Proteasome
Article Snippet: The antibodies used were as follows: anti-eIF4GI and anti-eIF4GII (gifts of Prof. Nahum Sonenberg); anti-DAP5 (CliniSciences #610742); anti-HA-7 (Sigma); anti-β-tubulin (GeneTex #6288022); anti-4E-BP1, anti-NRF2 and anti-p53 (Cell Signaling Technologies #9452, #12721, and #1C12, respectively); anti-Core 20S (Enzo Life Sciences #PW8155); and anti-NQO1 (Santa Cruz #C19).

Article Title: Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone‐responsive myopathy
Article Snippet: Reagents The following primary antibodies were used: anti‐p62 (SQSTM) (Abnova), anti‐LAMP2 (Abcam), anti‐FGF21 (abcam), anti‐Bip (BD Biosciences), anti‐Gapdh (Santa Cruz), anti‐SREBP1c (Santa Cruz), anti‐SREBP2 (abcam), anti‐ATF6 (abcam), anti‐LC3 (Nanotools), anti‐Tom20 (SantaCruz), anti‐lipin1 (SantaCruz, sc‐376874).

Incubation:

Article Title: Antioxidant Properties of Fullerene Derivatives Depend on Their Chemical Structure: A Study of Two Fullerene Derivatives on HELFs
Article Snippet: .. All investigated concentrations of VI-419-P3K cause an increase in the NRF2 expression within 3 h of incubation, but NRF2 does not translocate to the nucleus. .. In 24 h, the NRF2 expression increases both in the cytoplasm and the nucleus.

Article Title: Antioxidant Properties of Fullerene Derivatives Depend on Their Chemical Structure: A Study of Two Fullerene Derivatives on HELFs
Article Snippet: .. There was no translocation of NRF2 to the nucleus of HELFs after 3 h of incubation with any of the studied concentrations of GI-761 ( ). ..

Inhibition:

Article Title: Probing the Structural Requirements of Non-electrophilic Naphthalene-Based Nrf2 Activators
Article Snippet: .. Briefly, inhibition of the Keap1-Nrf2 interaction with small molecules was assayed using both the Kelch domain of Keap1 and an N-terminally fluorescein-labeled 9-mer peptide containing the ETGE motif derived from the Neh2 domain of Nrf2 [ ]. .. Sigmoidal concentration-response curves were fitted to the data using Graphpad Prism 6.1 software (see and and ).

Expressing:

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells
Article Snippet: .. Here, a significant reduction in MCM2 , Sox2 , and GFAP/nestin ( ) expressing NSPCs was noted in the DG of the Nrf2-/- mice. .. Moreover, the number of Dcx+ newborn neurons was also significantly reduced in the Nrf2-/- mice compared with WT controls ( ).

Western Blot:

Article Title: Loss of DJ-1 elicits retinal abnormalities, visual dysfunction, and increased oxidative stress in mice
Article Snippet: .. Immunoblots of retina/RPE lysates from 6 month-old mice revealed that DJ-1 KO l retinas displayed significant decreased immunoreactivity of red/green cone opsin, TH, Nrf2, ezrin and DJ-1 when compared to control lysates and normalized to the levels of GAPDH ( ). .. Quantification of these lysates ( ) demonstrated a 44% reduction in red/green opsin, a 40% reduction in TH, a 56% reduction in Nrf2 and a 83% reduction in ezrin signal intensity when comparing immunoreactivity in the control lysates.

Translocation Assay:

Article Title: Antioxidant Properties of Fullerene Derivatives Depend on Their Chemical Structure: A Study of Two Fullerene Derivatives on HELFs
Article Snippet: .. There was no translocation of NRF2 to the nucleus of HELFs after 3 h of incubation with any of the studied concentrations of GI-761 ( ). ..

Derivative Assay:

Article Title: Probing the Structural Requirements of Non-electrophilic Naphthalene-Based Nrf2 Activators
Article Snippet: .. Briefly, inhibition of the Keap1-Nrf2 interaction with small molecules was assayed using both the Kelch domain of Keap1 and an N-terminally fluorescein-labeled 9-mer peptide containing the ETGE motif derived from the Neh2 domain of Nrf2 [ ]. .. Sigmoidal concentration-response curves were fitted to the data using Graphpad Prism 6.1 software (see and and ).

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  • 93
    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: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Santa Cruz Biotechnology mouse anti human keap1 antibody
    <t>Keap1,</t> MCM3, and MCM-BP form a ternary complex. ( a ) Strep-Keap1 and FLAG-MCM3 pulldown experiments from Sf9 cells co-infected with baculoviruses expressing mouse MCM-BP together with WT or interaction deficient mutant MCM3 and Keap1 as indicated. Top panels show the Western blots of indicated proteins, bottom panel the blotted membranes that were stained with colloidal gold total protein stain. 1/300th of the starting extracts (‘input’) and 1/6th of the pulldown samples was loaded on each lane. See Supplementary Fig. S6 for full-length blots. ( b ) Strep-Keap1 - FLAG-MCM3 tandem affinity purification experiment from Sf9 cells co-infected with baculoviruses expressing all six mouse MCM2-7 subunits, Keap1, and MCM-BP. Coomassie brilliant blue stained SDS-PAGE gel on the left shows eluted material from both affinity purification steps, and unbound material from the FLAG affinity step in the middle lane. Resulting complexes were further resolved by Superose 6 size exclusion chromatography, the fractions of which are shown on right gel; co-elution of molecular weight markers is indicated at the bottom. The identity of protein bands was verified by mass spectrometry.
    Mouse Anti Human Keap1 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 79/100, 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