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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 "Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling"

Article Title: Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling

Journal: The FASEB Journal

doi: 10.1096/fj.201601155R

Effect of FPS-ZM1 on activation of DAMP signaling in PPE-induced experimental COPD.  A ) IKKα, IKKβ, and phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) were determined by Western blot analysis.  B ) JNK/ERK/p38 MAPK activation (phosphorylation) was determined by Western blot with the appropriate antibodies. The target protein bands were subjected to densitometric analysis normalized with β-actin.  C ) The canonical–noncanonical Rel/p65 NF-κB activation and MAPK activation and its correlation to Nrf2 translocation from the cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for the nuclear protein loading control, and β-actin was used for the cytoplasmic protein loading control. The corresponding density ratio was determined by the average intensity of the bands. Ctl, control group; ns, not significant. * P
Figure Legend Snippet: Effect of FPS-ZM1 on activation of DAMP signaling in PPE-induced experimental COPD. A ) IKKα, IKKβ, and phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) were determined by Western blot analysis. B ) JNK/ERK/p38 MAPK activation (phosphorylation) was determined by Western blot with the appropriate antibodies. The target protein bands were subjected to densitometric analysis normalized with β-actin. C ) The canonical–noncanonical Rel/p65 NF-κB activation and MAPK activation and its correlation to Nrf2 translocation from the cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for the nuclear protein loading control, and β-actin was used for the cytoplasmic protein loading control. The corresponding density ratio was determined by the average intensity of the bands. Ctl, control group; ns, not significant. * P

Techniques Used: Activation Assay, Western Blot, Translocation Assay, CTL Assay

Effect of FPS-ZM1 on CSE-induced activation of DAMP signaling in MLE-12 cells.  A – C ) After exposure to CSE in the presence or absence of FPS-ZM1 for 24 h, Western blot analysis was perfomed ( A ), and the expression levels of mRAGE in lung homogenates ( B ) and of circulating sRAGE ( C ) were determined by ELISA.  D ,  E ) IKKα, IKKβ, and the phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) ( D ) and the phosphorylation of JNK/ERK/p38 MAPK activation ( E ) were detected with the appropriate antibodies. β-Actin, total RelA/p65, total JNK1/2, total ERK1/2, and total p38 were used for loading controls.  F ) Correlation to Nrf2 translocation from cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for nuclear protein loading control, and β-actin was used for cytoplasmic protein loading control. The activation of DAMP signaling was inhibited by FPS-ZM1 treatment. Results are combined data from 3 independent experiments. Ctl, control group; ns, not significant. * P
Figure Legend Snippet: Effect of FPS-ZM1 on CSE-induced activation of DAMP signaling in MLE-12 cells. A – C ) After exposure to CSE in the presence or absence of FPS-ZM1 for 24 h, Western blot analysis was perfomed ( A ), and the expression levels of mRAGE in lung homogenates ( B ) and of circulating sRAGE ( C ) were determined by ELISA. D , E ) IKKα, IKKβ, and the phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) ( D ) and the phosphorylation of JNK/ERK/p38 MAPK activation ( E ) were detected with the appropriate antibodies. β-Actin, total RelA/p65, total JNK1/2, total ERK1/2, and total p38 were used for loading controls. F ) Correlation to Nrf2 translocation from cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for nuclear protein loading control, and β-actin was used for cytoplasmic protein loading control. The activation of DAMP signaling was inhibited by FPS-ZM1 treatment. Results are combined data from 3 independent experiments. Ctl, control group; ns, not significant. * P

Techniques Used: Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Translocation Assay, CTL Assay

3) Product Images from "A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling"

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

Journal: Nature

doi: 10.1038/s41586-018-0622-0

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

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

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

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

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

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

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

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

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

Techniques Used:

4) Product Images from "Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells"

Article Title: Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.0277

PI-induced Nrf2 activation suppresses PI-induced cell death (A) NCI-H157 cells were transiently transfected with control siRNAs or Nrf2 siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) for 24 h. Total cellular extracts were subjected to Western blot analysis for PARP, Nrf2, and GAPDH. (B) Cells were infected with adenovirus vectors expressing WT-Nrf2 or DN-Nrf2. After 48 h, the cells were stimulated with PS-341 (50 nM) for 24 h. Cell viability was determined by MTT assay. Data represent the mean ± SD of triplicate experiments. ** P
Figure Legend Snippet: PI-induced Nrf2 activation suppresses PI-induced cell death (A) NCI-H157 cells were transiently transfected with control siRNAs or Nrf2 siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) for 24 h. Total cellular extracts were subjected to Western blot analysis for PARP, Nrf2, and GAPDH. (B) Cells were infected with adenovirus vectors expressing WT-Nrf2 or DN-Nrf2. After 48 h, the cells were stimulated with PS-341 (50 nM) for 24 h. Cell viability was determined by MTT assay. Data represent the mean ± SD of triplicate experiments. ** P

Techniques Used: Activation Assay, Transfection, Western Blot, Infection, Expressing, MTT Assay

5) Product Images from "EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation"

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

Journal: Autophagy

doi: 10.1080/15548627.2018.1536530

EBV reduces mitochondrial biogenesis in differentiating monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) CYCS and (b) for NRF1 and TFAM expression by western blot. ACTB was used as a loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of CYCS:ACTB, NRF1:ACTB and TFAM:ACTB of 3 different experiments. * P value
Figure Legend Snippet: EBV reduces mitochondrial biogenesis in differentiating monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) CYCS and (b) for NRF1 and TFAM expression by western blot. ACTB was used as a loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of CYCS:ACTB, NRF1:ACTB and TFAM:ACTB of 3 different experiments. * P value

Techniques Used: Cell Culture, Expressing, Western Blot

6) Product Images from "Docosahexaenoic Acid Induces Expression of Heme Oxygenase-1 and NAD(P)H:quinone Oxidoreductase through Activation of Nrf2 in Human Mammary Epithelial Cells"

Article Title: Docosahexaenoic Acid Induces Expression of Heme Oxygenase-1 and NAD(P)H:quinone Oxidoreductase through Activation of Nrf2 in Human Mammary Epithelial Cells

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules22060969

Role of Nrf2 in DHA-induced expression of HO-1 and NQO1. ( A ) MCF-10A cells were transfected with siRNA control, siRNA Nrf2, or siRNA Nrf2 plus Nrf2 full-sequence vector for 12 h and exposed to DHA (25 μM) for another 9 h. Whole cell lysates were subjected to Western blot analysis. ( B ) Nrf2-WT or Nrf2-null MEF cells were incubated with 25 μM of DHA for 12 h, and the expression of Nrf2, HO-1, and NQO1 was measured by Western blot analysis. ( C ) MCF-10A cells were treated with DHA (25 μM) for 9 h and harvested to determine the ARE binding activity by the ChIP assay. Chromatin immunoprecipitated DNA was analyzed by RT-PCR with primers for distal E2 (−9.0 kb region) and E1 (−4.0 kb region) AREs as well as non-specific region (after −9.0 kb) of the HO-1 promoter.
Figure Legend Snippet: Role of Nrf2 in DHA-induced expression of HO-1 and NQO1. ( A ) MCF-10A cells were transfected with siRNA control, siRNA Nrf2, or siRNA Nrf2 plus Nrf2 full-sequence vector for 12 h and exposed to DHA (25 μM) for another 9 h. Whole cell lysates were subjected to Western blot analysis. ( B ) Nrf2-WT or Nrf2-null MEF cells were incubated with 25 μM of DHA for 12 h, and the expression of Nrf2, HO-1, and NQO1 was measured by Western blot analysis. ( C ) MCF-10A cells were treated with DHA (25 μM) for 9 h and harvested to determine the ARE binding activity by the ChIP assay. Chromatin immunoprecipitated DNA was analyzed by RT-PCR with primers for distal E2 (−9.0 kb region) and E1 (−4.0 kb region) AREs as well as non-specific region (after −9.0 kb) of the HO-1 promoter.

Techniques Used: Expressing, Transfection, Sequencing, Plasmid Preparation, Western Blot, Incubation, Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Role of ROS in DHA-induced Nrf2 activation mediated by PKCδ. ( A ) MCF-10A cells were treated with each indicated drug (EPA and DHA; 25 μM, NAC; 5 mM, H 2 O 2 ; 100 μM) or in proper combination for 3 h and then examined for the intracellular accumulation of ROS under the confocal microscope after DCF-DA fluorescence staining. ( B ) Cells were treated with DHA (25 μM) for the indicated time periods, and the effect of DHA on PKCδ activation was determined by Western blot analysis. ( C ) MCF-10A cells treated with DHA (25 μM) were harvested at the indicated time intervals. The effect of DHA on the phosphorylation of Ser 40 at Nrf2 was assessed by Western blot analysis. Each blot is a representative of three different experiments. Columns, means (n = 3); bars, SD. * p
Figure Legend Snippet: Role of ROS in DHA-induced Nrf2 activation mediated by PKCδ. ( A ) MCF-10A cells were treated with each indicated drug (EPA and DHA; 25 μM, NAC; 5 mM, H 2 O 2 ; 100 μM) or in proper combination for 3 h and then examined for the intracellular accumulation of ROS under the confocal microscope after DCF-DA fluorescence staining. ( B ) Cells were treated with DHA (25 μM) for the indicated time periods, and the effect of DHA on PKCδ activation was determined by Western blot analysis. ( C ) MCF-10A cells treated with DHA (25 μM) were harvested at the indicated time intervals. The effect of DHA on the phosphorylation of Ser 40 at Nrf2 was assessed by Western blot analysis. Each blot is a representative of three different experiments. Columns, means (n = 3); bars, SD. * p

Techniques Used: Activation Assay, Microscopy, Fluorescence, Staining, Western Blot

DHA-induced expression, nuclear translocation and transcriptional activity of Nrf2. ( A ) Total RNA was isolated from cells treated with or without DHA for indicated duration and analyzed by RT-PCR for detecting the level of Nrf2 mRNA; ( B ) MCF-10Acells were exposed to DHA (25 μM) were harvested at the indicated intervals, and the protein levels were assessed by Western blot analysis. ( C ) Nuclear extracts from MCF-10A cells were prepared at the indicated intervals after treatment with DHA (25 μM). ( D ) MCF-10A cells were treated with indicated concentrations of DHA for 9 h and the nuclear translocation of Nrf2 was assessed by Western blot analysis. ( E ) MCF-10A cells were incubated with DHA (25 μM) for 9 h and nuclear localization of Nrf2 was determined by immunocytochemical analysis. ( F ) MCF-10A cells were treated with DHA (25 μM) for 9 h after transfection with either an ARE luciferase construct or a control vector and analyzed for the Nrf2 transcriptional activity as described in Materials and Methods. Columns, means (n = 3); bars, SD. ***, p
Figure Legend Snippet: DHA-induced expression, nuclear translocation and transcriptional activity of Nrf2. ( A ) Total RNA was isolated from cells treated with or without DHA for indicated duration and analyzed by RT-PCR for detecting the level of Nrf2 mRNA; ( B ) MCF-10Acells were exposed to DHA (25 μM) were harvested at the indicated intervals, and the protein levels were assessed by Western blot analysis. ( C ) Nuclear extracts from MCF-10A cells were prepared at the indicated intervals after treatment with DHA (25 μM). ( D ) MCF-10A cells were treated with indicated concentrations of DHA for 9 h and the nuclear translocation of Nrf2 was assessed by Western blot analysis. ( E ) MCF-10A cells were incubated with DHA (25 μM) for 9 h and nuclear localization of Nrf2 was determined by immunocytochemical analysis. ( F ) MCF-10A cells were treated with DHA (25 μM) for 9 h after transfection with either an ARE luciferase construct or a control vector and analyzed for the Nrf2 transcriptional activity as described in Materials and Methods. Columns, means (n = 3); bars, SD. ***, p

Techniques Used: Expressing, Translocation Assay, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation, Transfection, Luciferase, Construct, Plasmid Preparation

7) Product Images from "Methylation of the KEAP1 gene promoter region in human colorectal cancer"

Article Title: Methylation of the KEAP1 gene promoter region in human colorectal cancer

Journal: BMC Cancer

doi: 10.1186/1471-2407-12-66

Expression of the Nrf2 target genes NQO- 1 and AKR1C1 after t-BHQ treatment . Real-time PCR analysis of the Nrf2 target genes NQO-1 ( A , left) and AKRC1 ( B , left) in HT29 cells (methylated) and Colo320DM cells (unmethylated). Cells were treated with the Keap1 stimulator t-BHQ for 24 h. Columns, mean ( n = 3); bars, SD. * P
Figure Legend Snippet: Expression of the Nrf2 target genes NQO- 1 and AKR1C1 after t-BHQ treatment . Real-time PCR analysis of the Nrf2 target genes NQO-1 ( A , left) and AKRC1 ( B , left) in HT29 cells (methylated) and Colo320DM cells (unmethylated). Cells were treated with the Keap1 stimulator t-BHQ for 24 h. Columns, mean ( n = 3); bars, SD. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Methylation

8) Product Images from "EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation"

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

Journal: Autophagy

doi: 10.1080/15548627.2018.1536530

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

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

9) Product Images from "Oxidative stress and cell damage in a model of precancerous lesions and advanced hepatocellular carcinoma in rats"

Article Title: Oxidative stress and cell damage in a model of precancerous lesions and advanced hepatocellular carcinoma in rats

Journal: Toxicology Reports

doi: 10.1016/j.toxrep.2014.11.015

Western blot analysis of TGF-1β, eNOS, and iNOS. Protein from liver extracts was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. (A) Representative images. (B) Densitometric quantification. Values are expressed as means ± SD ( n = 5). a p
Figure Legend Snippet: Western blot analysis of TGF-1β, eNOS, and iNOS. Protein from liver extracts was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. (A) Representative images. (B) Densitometric quantification. Values are expressed as means ± SD ( n = 5). a p

Techniques Used: Western Blot, Polyacrylamide Gel Electrophoresis

10) Product Images from "Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels"

Article Title: Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels

Journal: Scientific Reports

doi: 10.1038/s41598-018-26049-5

Effect of plasma on chicken sperm mRNA and protein expression. Semen of 60-week-old cocks was exposed to varying plasma potentials for 20 s. Relative mRNA levels of the following genes were measured: ( a ) NOX4 , NRF2 , and KEAP1 ; ( b ) SOD , CAT , and GPx ; ( c ) PRDX1 , PRDX 3, PRDX4 , and PRDX6 ; ( d ) ATP5A1 , ATP5B , ATP5C1 , ATP5F1 , ATP5G1 , ATP5G3 , ATP5H , ATP5I , ATP5J , ATP5J 2, ATP5L , and ATP5S ; and ( e ) AMPKα2 , AMPKβ2 , AMPKγ3 , and mTOR . ( f . The grouping of gels/blots cropped from different gels. All blots were visualized with 5 min exposure time. Relative protein levels of ( g ) NRF2, KEAP1, PRDX4, ( h ) ATP5A, ( i ) p-AMPKα/AMPKα, and ( j ) p-mTOR/mTOR. Values are expressed as the mean ± standard error (n = 3) of three replicates; n represents an individual cock. * p
Figure Legend Snippet: Effect of plasma on chicken sperm mRNA and protein expression. Semen of 60-week-old cocks was exposed to varying plasma potentials for 20 s. Relative mRNA levels of the following genes were measured: ( a ) NOX4 , NRF2 , and KEAP1 ; ( b ) SOD , CAT , and GPx ; ( c ) PRDX1 , PRDX 3, PRDX4 , and PRDX6 ; ( d ) ATP5A1 , ATP5B , ATP5C1 , ATP5F1 , ATP5G1 , ATP5G3 , ATP5H , ATP5I , ATP5J , ATP5J 2, ATP5L , and ATP5S ; and ( e ) AMPKα2 , AMPKβ2 , AMPKγ3 , and mTOR . ( f . The grouping of gels/blots cropped from different gels. All blots were visualized with 5 min exposure time. Relative protein levels of ( g ) NRF2, KEAP1, PRDX4, ( h ) ATP5A, ( i ) p-AMPKα/AMPKα, and ( j ) p-mTOR/mTOR. Values are expressed as the mean ± standard error (n = 3) of three replicates; n represents an individual cock. * p

Techniques Used: Expressing

11) Product Images from "Sulforaphane Protects the Liver against CdSe Quantum Dot-Induced Cytotoxicity"

Article Title: Sulforaphane Protects the Liver against CdSe Quantum Dot-Induced Cytotoxicity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0138771

Effect of knockdown TR-1, Keap1 and Nrf2 on cytotoxicity in HHL-5 cells exposed to CdSe QDs. TR-1, Keap1 or Nrf2 were knocked down, respectively. Allstars (AS) were used as a negative control. Cells were incubated with 5 μM SFN or DMSO (0.1%) control for 24 h then exposed to 20 μM CdSe QDs for 24 h. Cell viability was measured by MTT assay, and data shown as means ± SD (n = 6). Significant levels (*P
Figure Legend Snippet: Effect of knockdown TR-1, Keap1 and Nrf2 on cytotoxicity in HHL-5 cells exposed to CdSe QDs. TR-1, Keap1 or Nrf2 were knocked down, respectively. Allstars (AS) were used as a negative control. Cells were incubated with 5 μM SFN or DMSO (0.1%) control for 24 h then exposed to 20 μM CdSe QDs for 24 h. Cell viability was measured by MTT assay, and data shown as means ± SD (n = 6). Significant levels (*P

Techniques Used: Negative Control, Incubation, MTT Assay

12) Product Images from "A novel cystine based antioxidant attenuates oxidative stress and hepatic steatosis in diet-induced obese mice"

Article Title: A novel cystine based antioxidant attenuates oxidative stress and hepatic steatosis in diet-induced obese mice

Journal: Experimental and molecular pathology

doi: 10.1016/j.yexmp.2011.04.009

F1 treatment prevents activation of JNK and p38 MAPK, perturbation of BAX/BCL-2 ratio, and caspase activation in livers in mice fed with HFD. A: EIA revels significantly (P
Figure Legend Snippet: F1 treatment prevents activation of JNK and p38 MAPK, perturbation of BAX/BCL-2 ratio, and caspase activation in livers in mice fed with HFD. A: EIA revels significantly (P

Techniques Used: Activation Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

13) Product Images from "Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling"

Article Title: Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling

Journal: The FASEB Journal

doi: 10.1096/fj.201601155R

Effect of FPS-ZM1 on activation of DAMP signaling in PPE-induced experimental COPD. A ) IKKα, IKKβ, and phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) were determined by Western blot analysis. B ) JNK/ERK/p38 MAPK activation (phosphorylation) was determined by Western blot with the appropriate antibodies. The target protein bands were subjected to densitometric analysis normalized with β-actin. C ) The canonical–noncanonical Rel/p65 NF-κB activation and MAPK activation and its correlation to Nrf2 translocation from the cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for the nuclear protein loading control, and β-actin was used for the cytoplasmic protein loading control. The corresponding density ratio was determined by the average intensity of the bands. Ctl, control group; ns, not significant. * P
Figure Legend Snippet: Effect of FPS-ZM1 on activation of DAMP signaling in PPE-induced experimental COPD. A ) IKKα, IKKβ, and phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) were determined by Western blot analysis. B ) JNK/ERK/p38 MAPK activation (phosphorylation) was determined by Western blot with the appropriate antibodies. The target protein bands were subjected to densitometric analysis normalized with β-actin. C ) The canonical–noncanonical Rel/p65 NF-κB activation and MAPK activation and its correlation to Nrf2 translocation from the cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for the nuclear protein loading control, and β-actin was used for the cytoplasmic protein loading control. The corresponding density ratio was determined by the average intensity of the bands. Ctl, control group; ns, not significant. * P

Techniques Used: Activation Assay, Western Blot, Translocation Assay, CTL Assay

Effect of FPS-ZM1 on CSE-induced activation of DAMP signaling in MLE-12 cells. A – C ) After exposure to CSE in the presence or absence of FPS-ZM1 for 24 h, Western blot analysis was perfomed ( A ), and the expression levels of mRAGE in lung homogenates ( B ) and of circulating sRAGE ( C ) were determined by ELISA. D , E ) IKKα, IKKβ, and the phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) ( D ) and the phosphorylation of JNK/ERK/p38 MAPK activation ( E ) were detected with the appropriate antibodies. β-Actin, total RelA/p65, total JNK1/2, total ERK1/2, and total p38 were used for loading controls. F ) Correlation to Nrf2 translocation from cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for nuclear protein loading control, and β-actin was used for cytoplasmic protein loading control. The activation of DAMP signaling was inhibited by FPS-ZM1 treatment. Results are combined data from 3 independent experiments. Ctl, control group; ns, not significant. * P
Figure Legend Snippet: Effect of FPS-ZM1 on CSE-induced activation of DAMP signaling in MLE-12 cells. A – C ) After exposure to CSE in the presence or absence of FPS-ZM1 for 24 h, Western blot analysis was perfomed ( A ), and the expression levels of mRAGE in lung homogenates ( B ) and of circulating sRAGE ( C ) were determined by ELISA. D , E ) IKKα, IKKβ, and the phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) ( D ) and the phosphorylation of JNK/ERK/p38 MAPK activation ( E ) were detected with the appropriate antibodies. β-Actin, total RelA/p65, total JNK1/2, total ERK1/2, and total p38 were used for loading controls. F ) Correlation to Nrf2 translocation from cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for nuclear protein loading control, and β-actin was used for cytoplasmic protein loading control. The activation of DAMP signaling was inhibited by FPS-ZM1 treatment. Results are combined data from 3 independent experiments. Ctl, control group; ns, not significant. * P

Techniques Used: Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Translocation Assay, CTL Assay

14) Product Images from "Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells"

Article Title: Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.0277

PI-induced Nrf2 activation suppresses PI-induced cell death (A) NCI-H157 cells were transiently transfected with control siRNAs or Nrf2 siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) for 24 h. Total cellular extracts were subjected to Western blot analysis for PARP, Nrf2, and GAPDH. (B) Cells were infected with adenovirus vectors expressing WT-Nrf2 or DN-Nrf2. After 48 h, the cells were stimulated with PS-341 (50 nM) for 24 h. Cell viability was determined by MTT assay. Data represent the mean ± SD of triplicate experiments. ** P
Figure Legend Snippet: PI-induced Nrf2 activation suppresses PI-induced cell death (A) NCI-H157 cells were transiently transfected with control siRNAs or Nrf2 siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) for 24 h. Total cellular extracts were subjected to Western blot analysis for PARP, Nrf2, and GAPDH. (B) Cells were infected with adenovirus vectors expressing WT-Nrf2 or DN-Nrf2. After 48 h, the cells were stimulated with PS-341 (50 nM) for 24 h. Cell viability was determined by MTT assay. Data represent the mean ± SD of triplicate experiments. ** P

Techniques Used: Activation Assay, Transfection, Western Blot, Infection, Expressing, MTT Assay

15) Product Images from "A specific expression profile of LC3B and p62 is associated with nonresponse to neoadjuvant chemotherapy in esophageal adenocarcinomas"

Article Title: A specific expression profile of LC3B and p62 is associated with nonresponse to neoadjuvant chemotherapy in esophageal adenocarcinomas

Journal: PLoS ONE

doi: 10.1371/journal.pone.0197610

Examples of immunohistochemical stainings. (a) High LC3B dot like staining (score 2). (b) Low LC3B dot like staining (score 1), note a small nerve serving as internal positive control. (c) High p62 cytoplasmic staining (score 3), while negative nuclear staining. (d) Low p62 cytoplasmic/dot-like staining (scores 0), positive nuclear staining. (e) High cytoplasmic (score 2) and low dot-like (score 1) p62 staining. (f) Low cytoplasmic (score 1) and high dot like (score 2) p62 staining. 40x magnification for all images. Error bars indicate 20µm.
Figure Legend Snippet: Examples of immunohistochemical stainings. (a) High LC3B dot like staining (score 2). (b) Low LC3B dot like staining (score 1), note a small nerve serving as internal positive control. (c) High p62 cytoplasmic staining (score 3), while negative nuclear staining. (d) Low p62 cytoplasmic/dot-like staining (scores 0), positive nuclear staining. (e) High cytoplasmic (score 2) and low dot-like (score 1) p62 staining. (f) Low cytoplasmic (score 1) and high dot like (score 2) p62 staining. 40x magnification for all images. Error bars indicate 20µm.

Techniques Used: Immunohistochemistry, Staining, Positive Control

Cytoplasmic expression of p62 results in decreased responsiveness of EAC cells to paclitaxel. (a) OE19 p62 knockdown cells were transiently transfected with either a cytoplasmic or nuclear GFP-tagged p62 expression plasmid. GFP (GFP-p62 fusion proteins) and nuclear DAPI staining as analyzed by confocal microscopy are shown. (b) Annexin V/DAPI fluorescence-activated cell sorting (FACS) analysis of OE19 cells expressing cytoplasmic or nuclear p62 after 48 h of paclitaxel treatment. Bars represent four experimental replicates.
Figure Legend Snippet: Cytoplasmic expression of p62 results in decreased responsiveness of EAC cells to paclitaxel. (a) OE19 p62 knockdown cells were transiently transfected with either a cytoplasmic or nuclear GFP-tagged p62 expression plasmid. GFP (GFP-p62 fusion proteins) and nuclear DAPI staining as analyzed by confocal microscopy are shown. (b) Annexin V/DAPI fluorescence-activated cell sorting (FACS) analysis of OE19 cells expressing cytoplasmic or nuclear p62 after 48 h of paclitaxel treatment. Bars represent four experimental replicates.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Fluorescence, FACS

16) Product Images from "Nrf2-dysregulation correlates with reduced synthesis and low glutathione levels in experimental autoimmune encephalomyelitis"

Article Title: Nrf2-dysregulation correlates with reduced synthesis and low glutathione levels in experimental autoimmune encephalomyelitis

Journal: Journal of neurochemistry

doi: 10.1111/jnc.13837

GCLc and GSS mRNA and protein levels are reduced during the course of EAE. (a, b) GCLc ( Gclc ) and GSS ( Gss and are expressed relative to the geometric mean of 4 reference genes as described in “Materials and Methods”. Values represent the mean ± SEM of 3–8 animals per experimental group. (c, d) GCLc and GSS protein levels in the spinal cord of control and EAE mice were determined by western blot analysis as described under “Materials and Methods” and are expressed relative to those of GAPDH. Values represent the mean ± SEM of 3–7 animals per experimental group. Asterisks denote values that are statistically different ( p
Figure Legend Snippet: GCLc and GSS mRNA and protein levels are reduced during the course of EAE. (a, b) GCLc ( Gclc ) and GSS ( Gss and are expressed relative to the geometric mean of 4 reference genes as described in “Materials and Methods”. Values represent the mean ± SEM of 3–8 animals per experimental group. (c, d) GCLc and GSS protein levels in the spinal cord of control and EAE mice were determined by western blot analysis as described under “Materials and Methods” and are expressed relative to those of GAPDH. Values represent the mean ± SEM of 3–7 animals per experimental group. Asterisks denote values that are statistically different ( p

Techniques Used: Mouse Assay, Western Blot

γ-GT and xCT mRNA and protein levels are diminished during the course of EAE. (a, b) γ-GT ( Ggt1 ) and xCT ( Slc7a11 and are expressed relative to the geometric mean of 4 reference genes as described in “Materials and Methods”. Values represent the mean ± SEM of 3–8 animals per experimental group. (c, d) γ-GT and xCT protein levels in the spinal cord of control and EAE mice were determined by western blot analysis as described under “Materials and Methods” and are expressed relative to those of GAPDH. Values represent the mean ± SEM of 3–7 animals per experimental group. Asterisks denote values that are statistically different ( p
Figure Legend Snippet: γ-GT and xCT mRNA and protein levels are diminished during the course of EAE. (a, b) γ-GT ( Ggt1 ) and xCT ( Slc7a11 and are expressed relative to the geometric mean of 4 reference genes as described in “Materials and Methods”. Values represent the mean ± SEM of 3–8 animals per experimental group. (c, d) γ-GT and xCT protein levels in the spinal cord of control and EAE mice were determined by western blot analysis as described under “Materials and Methods” and are expressed relative to those of GAPDH. Values represent the mean ± SEM of 3–7 animals per experimental group. Asterisks denote values that are statistically different ( p

Techniques Used: Mouse Assay, Western Blot

17) Product Images from "Nrf2 induces cisplatin resistance through activation of autophagy in ovarian carcinoma"

Article Title: Nrf2 induces cisplatin resistance through activation of autophagy in ovarian carcinoma

Journal: International Journal of Clinical and Experimental Pathology

doi:

Effects of Nrf2 knockdown on cisplatin sensitivity in resistant A2780cp cells. A. After cells were transfected with Nrf2 siRNA (100 nM) for 48 h, cell lysates were collected for western blot analyses for Nrf2, Keap1, NQO1 and HO-1. B. Cells were transfected
Figure Legend Snippet: Effects of Nrf2 knockdown on cisplatin sensitivity in resistant A2780cp cells. A. After cells were transfected with Nrf2 siRNA (100 nM) for 48 h, cell lysates were collected for western blot analyses for Nrf2, Keap1, NQO1 and HO-1. B. Cells were transfected

Techniques Used: Transfection, Western Blot

18) Product Images from "Reduced mammalian target of rapamycin activity facilitates mitochondrial retrograde signaling and increases life span in normal human fibroblasts"

Article Title: Reduced mammalian target of rapamycin activity facilitates mitochondrial retrograde signaling and increases life span in normal human fibroblasts

Journal: Aging cell

doi: 10.1111/acel.12122

Inhibition of mammalian target of rapamycin (mTOR) increases the levels of proteins associated with mitochondrial biogenesis and activation of NFE2L2. (A) Representative Western blots of the steady-state levels of PGC-1α, TFAm, NFE2L2, NRF1, and
Figure Legend Snippet: Inhibition of mammalian target of rapamycin (mTOR) increases the levels of proteins associated with mitochondrial biogenesis and activation of NFE2L2. (A) Representative Western blots of the steady-state levels of PGC-1α, TFAm, NFE2L2, NRF1, and

Techniques Used: Inhibition, Activation Assay, Western Blot, Pyrolysis Gas Chromatography

19) Product Images from "Sulforaphane Protects the Liver against CdSe Quantum Dot-Induced Cytotoxicity"

Article Title: Sulforaphane Protects the Liver against CdSe Quantum Dot-Induced Cytotoxicity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0138771

Effect of knockdown TR-1, Keap1 and Nrf2 on cytotoxicity in HHL-5 cells exposed to CdSe QDs. TR-1, Keap1 or Nrf2 were knocked down, respectively. Allstars (AS) were used as a negative control. Cells were incubated with 5 μM SFN or DMSO (0.1%) control for 24 h then exposed to 20 μM CdSe QDs for 24 h. Cell viability was measured by MTT assay, and data shown as means ± SD (n = 6). Significant levels (*P
Figure Legend Snippet: Effect of knockdown TR-1, Keap1 and Nrf2 on cytotoxicity in HHL-5 cells exposed to CdSe QDs. TR-1, Keap1 or Nrf2 were knocked down, respectively. Allstars (AS) were used as a negative control. Cells were incubated with 5 μM SFN or DMSO (0.1%) control for 24 h then exposed to 20 μM CdSe QDs for 24 h. Cell viability was measured by MTT assay, and data shown as means ± SD (n = 6). Significant levels (*P

Techniques Used: Negative Control, Incubation, MTT Assay

20) Product Images from "Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels"

Article Title: Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels

Journal: Scientific Reports

doi: 10.1038/s41598-018-26049-5

Effect of plasma on chicken sperm mRNA and protein expression. Semen of 60-week-old cocks was exposed to varying plasma potentials for 20 s. Relative mRNA levels of the following genes were measured: ( a ) NOX4 , NRF2 , and KEAP1 ; ( b ) SOD , CAT , and GPx ; ( c ) PRDX1 , PRDX 3, PRDX4 , and PRDX6 ; ( d ) ATP5A1 , ATP5B , ATP5C1 , ATP5F1 , ATP5G1 , ATP5G3 , ATP5H , ATP5I , ATP5J , ATP5J 2, ATP5L , and ATP5S ; and ( e ) AMPKα2 , AMPKβ2 , AMPKγ3 , and mTOR . ( f ) Western blot analysis of protein bands. Uncropped immunoblot scans are presented in Supplementary Figure S1 . The grouping of gels/blots cropped from different gels. All blots were visualized with 5 min exposure time. Relative protein levels of ( g ) NRF2, KEAP1, PRDX4, ( h ) ATP5A, ( i ) p-AMPKα/AMPKα, and ( j ) p-mTOR/mTOR. Values are expressed as the mean ± standard error (n = 3) of three replicates; n represents an individual cock. * p
Figure Legend Snippet: Effect of plasma on chicken sperm mRNA and protein expression. Semen of 60-week-old cocks was exposed to varying plasma potentials for 20 s. Relative mRNA levels of the following genes were measured: ( a ) NOX4 , NRF2 , and KEAP1 ; ( b ) SOD , CAT , and GPx ; ( c ) PRDX1 , PRDX 3, PRDX4 , and PRDX6 ; ( d ) ATP5A1 , ATP5B , ATP5C1 , ATP5F1 , ATP5G1 , ATP5G3 , ATP5H , ATP5I , ATP5J , ATP5J 2, ATP5L , and ATP5S ; and ( e ) AMPKα2 , AMPKβ2 , AMPKγ3 , and mTOR . ( f ) Western blot analysis of protein bands. Uncropped immunoblot scans are presented in Supplementary Figure S1 . The grouping of gels/blots cropped from different gels. All blots were visualized with 5 min exposure time. Relative protein levels of ( g ) NRF2, KEAP1, PRDX4, ( h ) ATP5A, ( i ) p-AMPKα/AMPKα, and ( j ) p-mTOR/mTOR. Values are expressed as the mean ± standard error (n = 3) of three replicates; n represents an individual cock. * p

Techniques Used: Expressing, Western Blot

Cytosine methylation analysis of chicken spermatozoa. Cytosine methylation of ( a ) NRF 2, ( b ) KEAP1 , ( c ) PRDX4 , ( d ) ATP5A1 , ( e ) AMPKα2 , and ( f ) mTOR were analyzed using CyMATE. Lengths of sequenced regions and position of cytosine are shown schematically. The order of the individual sequences of ten clones is listed on the left. 0 represents the control group; 11.7 represents plasma-treated group at 11.7 kV for 20 s; and 27.6 represents plasma-treated group at 27.6 kV for 20 s. The reference sequence is shown in the first line. The sequence is distinguished by circles for CG, squares for CHG, and triangles for CHH. Filled symbols represent methylated cytosine, and open symbols represent unmethylated cytosine. Average methylation levels for CG, CHG, and CHH of ( g ) NRF2 , ( h ) KEAP1 , ( i ) PRDX4 , ( j ) ATP5A1 , ( k ) AMPKα2 , and ( l ) mTOR .
Figure Legend Snippet: Cytosine methylation analysis of chicken spermatozoa. Cytosine methylation of ( a ) NRF 2, ( b ) KEAP1 , ( c ) PRDX4 , ( d ) ATP5A1 , ( e ) AMPKα2 , and ( f ) mTOR were analyzed using CyMATE. Lengths of sequenced regions and position of cytosine are shown schematically. The order of the individual sequences of ten clones is listed on the left. 0 represents the control group; 11.7 represents plasma-treated group at 11.7 kV for 20 s; and 27.6 represents plasma-treated group at 27.6 kV for 20 s. The reference sequence is shown in the first line. The sequence is distinguished by circles for CG, squares for CHG, and triangles for CHH. Filled symbols represent methylated cytosine, and open symbols represent unmethylated cytosine. Average methylation levels for CG, CHG, and CHH of ( g ) NRF2 , ( h ) KEAP1 , ( i ) PRDX4 , ( j ) ATP5A1 , ( k ) AMPKα2 , and ( l ) mTOR .

Techniques Used: Methylation, Clone Assay, Sequencing

21) Product Images from "Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis"

Article Title: Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq212

Nrf2 genome-wide ChIP-Seq dataset. ( A ) Keap1 −/− MEFs have constitutive transcriptional activation of Nrf2 target genes (i) Nuclear Nrf2 protein levels in Keap1 −/− , WT and Nrf2 −/− MEFs using immunoblot analysis, (ii) ChIP–PCR and densitometry quantification analysis for known Nrf2-binding site in promoter of Nqo1 and Gsta3, (iii) qRT-PCR analysis of known Nrf2 targets, Nqo1 and Gsta3 and (iv) Immunoblot analysis for Nqo1 and Gsta3 in the MEFs. β-Actin was used as the loading control. ( B ) The distribution of Nrf2 ChIP-Seq peaks relative to the closest gene transcription start sites is similar to background. ( C ) Nrf2 ChIP-Seq peaks are enriched for high-scoring Nrf2 predicted motifs compared with background. ( D ) High-scoring predicted sites are preferentially located near the peak maximum height in the ChIP-Seq peaks whereas they are uniformly distributed in the background sequences. * P -value
Figure Legend Snippet: Nrf2 genome-wide ChIP-Seq dataset. ( A ) Keap1 −/− MEFs have constitutive transcriptional activation of Nrf2 target genes (i) Nuclear Nrf2 protein levels in Keap1 −/− , WT and Nrf2 −/− MEFs using immunoblot analysis, (ii) ChIP–PCR and densitometry quantification analysis for known Nrf2-binding site in promoter of Nqo1 and Gsta3, (iii) qRT-PCR analysis of known Nrf2 targets, Nqo1 and Gsta3 and (iv) Immunoblot analysis for Nqo1 and Gsta3 in the MEFs. β-Actin was used as the loading control. ( B ) The distribution of Nrf2 ChIP-Seq peaks relative to the closest gene transcription start sites is similar to background. ( C ) Nrf2 ChIP-Seq peaks are enriched for high-scoring Nrf2 predicted motifs compared with background. ( D ) High-scoring predicted sites are preferentially located near the peak maximum height in the ChIP-Seq peaks whereas they are uniformly distributed in the background sequences. * P -value

Techniques Used: Genome Wide, Chromatin Immunoprecipitation, Activation Assay, Polymerase Chain Reaction, Binding Assay, Quantitative RT-PCR

Antioxidant and xenobiotic detoxification genes are validated as Nrf2 direct targets. ( A) and (B ) The panels represent ChIP–PCR analysis and densitometry quantification for selected known Nrf2 target gene-binding sites—Txnrd1_P1 and its novel binding site Txnrd1_P2, novel binding sites in Gsta4, three Gstm1-binding sites (Gstm1_P1, Gstm1_P2, Gstm1_P3), Gstm3, Srxn1, Ephx1 and Als2. ( C ) qRT-PCR mRNA expression analysis in MEFs. (D ) Immunoblot analysis for Txnrd1, Gsta4, Gstm1, Srxn1 and Als2 in MEFs. β-Actin was used as the loading control. ( E ) qRT-PCR for mRNA expression in lung lysates from WT and Nrf2 −/− Air and CS exposed mice, corroborating with the in vitro Nrf2 targets. * P -value
Figure Legend Snippet: Antioxidant and xenobiotic detoxification genes are validated as Nrf2 direct targets. ( A) and (B ) The panels represent ChIP–PCR analysis and densitometry quantification for selected known Nrf2 target gene-binding sites—Txnrd1_P1 and its novel binding site Txnrd1_P2, novel binding sites in Gsta4, three Gstm1-binding sites (Gstm1_P1, Gstm1_P2, Gstm1_P3), Gstm3, Srxn1, Ephx1 and Als2. ( C ) qRT-PCR mRNA expression analysis in MEFs. (D ) Immunoblot analysis for Txnrd1, Gsta4, Gstm1, Srxn1 and Als2 in MEFs. β-Actin was used as the loading control. ( E ) qRT-PCR for mRNA expression in lung lysates from WT and Nrf2 −/− Air and CS exposed mice, corroborating with the in vitro Nrf2 targets. * P -value

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Quantitative RT-PCR, Expressing, Mouse Assay, In Vitro

Cell proliferation genes and cell-cycle regulators are direct transcriptional targets of Nrf2. ( A) and ( B) ChIP–PCR and densitometry quantification analysis for cell proliferation genes and Cdkn genes—Cdkn1a, Cdkn2b-binding site (Cdkn2b_P1), comparing Keap1 −/− , WT and Nrf2 −/− MEFs. ( C ) qRT-PCR for mRNA expression analysis of proliferation genes and Cdkns respectively. ( D ) Immunoblot analysis for Bmpr1a, Igf1, Npnt, Pdgfc, Vegfc CDKN1A and CDKN2A in MEFs. β-Actin was used as the loading control. ( E ) Quantos TM Cell proliferation assay. ( F ) BrdU chemiluminescence assay. ( G) Caspase activity (apoptosis) assay. ( H ) Cell senescence assay. ( I ) qRT-PCR for mRNA expression in lung lysates from WT and Nrf2 −/− Air and CS exposed mice corroborating with the in vitro Nrf2 targets. * P -value
Figure Legend Snippet: Cell proliferation genes and cell-cycle regulators are direct transcriptional targets of Nrf2. ( A) and ( B) ChIP–PCR and densitometry quantification analysis for cell proliferation genes and Cdkn genes—Cdkn1a, Cdkn2b-binding site (Cdkn2b_P1), comparing Keap1 −/− , WT and Nrf2 −/− MEFs. ( C ) qRT-PCR for mRNA expression analysis of proliferation genes and Cdkns respectively. ( D ) Immunoblot analysis for Bmpr1a, Igf1, Npnt, Pdgfc, Vegfc CDKN1A and CDKN2A in MEFs. β-Actin was used as the loading control. ( E ) Quantos TM Cell proliferation assay. ( F ) BrdU chemiluminescence assay. ( G) Caspase activity (apoptosis) assay. ( H ) Cell senescence assay. ( I ) qRT-PCR for mRNA expression in lung lysates from WT and Nrf2 −/− Air and CS exposed mice corroborating with the in vitro Nrf2 targets. * P -value

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Quantitative RT-PCR, Expressing, Proliferation Assay, Chemiluminescence Immunoassay, Activity Assay, Apoptosis Assay, Mouse Assay, In Vitro

Global transcriptional profiling of MEF genetic models to decipher comprehensive Nrf2-dependent transcriptional targets. ( A ) Scheme depicts definitions for classification of Nrf2 target genes as inducible and/or basally regulated by Nrf2. Basal targets: differentially expressed genes between WT versus Nrf2 −/− MEFs; Inducible targets: differentially expressed genes between Keap1 −/− versus WT MEFs; and Common targets: genes satisfying both the former comparison criteria. ( B ) The panel represents the overlap of differentially expressed genes across experiments. The orange circle represents genes that were downregulated in Nrf2 −/− MEFs compared with WT (basal targets) while the blue circle shows the genes upregulated in Keap1 −/− MEFs compared with WT (inducible targets). ( C ) The panel represents the overlap between the microarray datasets and the ChIP-Seq dataset and intersections represent the genes common in the two datasets to identify Nrf2 direct (basal, inducible and common) transcriptional targets.
Figure Legend Snippet: Global transcriptional profiling of MEF genetic models to decipher comprehensive Nrf2-dependent transcriptional targets. ( A ) Scheme depicts definitions for classification of Nrf2 target genes as inducible and/or basally regulated by Nrf2. Basal targets: differentially expressed genes between WT versus Nrf2 −/− MEFs; Inducible targets: differentially expressed genes between Keap1 −/− versus WT MEFs; and Common targets: genes satisfying both the former comparison criteria. ( B ) The panel represents the overlap of differentially expressed genes across experiments. The orange circle represents genes that were downregulated in Nrf2 −/− MEFs compared with WT (basal targets) while the blue circle shows the genes upregulated in Keap1 −/− MEFs compared with WT (inducible targets). ( C ) The panel represents the overlap between the microarray datasets and the ChIP-Seq dataset and intersections represent the genes common in the two datasets to identify Nrf2 direct (basal, inducible and common) transcriptional targets.

Techniques Used: Microarray, Chromatin Immunoprecipitation

Nrf2-binding profiles. ( A ) The original Nrf2-binding profile was defined by the alignment of the 20 known binding sites described in Table 1 . ( B ) The motif generated by the MEME motif discovery algorithm ( 50 ) on the Nrf2 ChIP-Seq dataset is highly similar to the original profile with a higher information content (IC; in bits). The position Frequency Matrix (PFM) captures the count of each nucleotide found at each position in the motif and the logo provides a visualization of the data. The information content is the sum of the information content of each position and is computed as described previously ( 61 ).
Figure Legend Snippet: Nrf2-binding profiles. ( A ) The original Nrf2-binding profile was defined by the alignment of the 20 known binding sites described in Table 1 . ( B ) The motif generated by the MEME motif discovery algorithm ( 50 ) on the Nrf2 ChIP-Seq dataset is highly similar to the original profile with a higher information content (IC; in bits). The position Frequency Matrix (PFM) captures the count of each nucleotide found at each position in the motif and the logo provides a visualization of the data. The information content is the sum of the information content of each position and is computed as described previously ( 61 ).

Techniques Used: Binding Assay, Generated, Chromatin Immunoprecipitation

Summary of Nrf2-regulated global transcriptomics circuit. This Nrf2 interaction network was generated using the String interaction network analysis tool ( http://string.embl.de ). The genes represented are the basal (black), inducible (white) and common (grey) target genes associated with cell proliferation and/or glutathione metabolism as defined by the functional clusters obtained through the DAVID resource and described in Table 3 .
Figure Legend Snippet: Summary of Nrf2-regulated global transcriptomics circuit. This Nrf2 interaction network was generated using the String interaction network analysis tool ( http://string.embl.de ). The genes represented are the basal (black), inducible (white) and common (grey) target genes associated with cell proliferation and/or glutathione metabolism as defined by the functional clusters obtained through the DAVID resource and described in Table 3 .

Techniques Used: Generated, Functional Assay

22) Product Images from "Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells"

Article Title: Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.0277

Macroautophagy mediates PI-induced IκBα degradation (A, B) NCI-H157 cells were transiently transfected with control shRNAs, Lamp2 shRNAs, control siRNAs, or LC3B siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) or MG132 (20 μM) for 8 h. Total cellular extracts were subjected to Western blot analysis for IκBα, Lamp2a, Lamp2, LC3B, and GAPDH. Results are representative of three independent experiments.
Figure Legend Snippet: Macroautophagy mediates PI-induced IκBα degradation (A, B) NCI-H157 cells were transiently transfected with control shRNAs, Lamp2 shRNAs, control siRNAs, or LC3B siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) or MG132 (20 μM) for 8 h. Total cellular extracts were subjected to Western blot analysis for IκBα, Lamp2a, Lamp2, LC3B, and GAPDH. Results are representative of three independent experiments.

Techniques Used: Transfection, Western Blot

PI-induced Nrf2 activation suppresses PI-induced cell death (A) NCI-H157 cells were transiently transfected with control siRNAs or Nrf2 siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) for 24 h. Total cellular extracts were subjected to Western blot analysis for PARP, Nrf2, and GAPDH. (B) Cells were infected with adenovirus vectors expressing WT-Nrf2 or DN-Nrf2. After 48 h, the cells were stimulated with PS-341 (50 nM) for 24 h. Cell viability was determined by MTT assay. Data represent the mean ± SD of triplicate experiments. ** P
Figure Legend Snippet: PI-induced Nrf2 activation suppresses PI-induced cell death (A) NCI-H157 cells were transiently transfected with control siRNAs or Nrf2 siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) for 24 h. Total cellular extracts were subjected to Western blot analysis for PARP, Nrf2, and GAPDH. (B) Cells were infected with adenovirus vectors expressing WT-Nrf2 or DN-Nrf2. After 48 h, the cells were stimulated with PS-341 (50 nM) for 24 h. Cell viability was determined by MTT assay. Data represent the mean ± SD of triplicate experiments. ** P

Techniques Used: Activation Assay, Transfection, Western Blot, Infection, Expressing, MTT Assay

23) Product Images from "The Cinnamon-derived Dietary Factor Cinnamic Aldehyde Activates the Nrf2-dependent Antioxidant Response in Human Epithelial Colon Cells"

Article Title: The Cinnamon-derived Dietary Factor Cinnamic Aldehyde Activates the Nrf2-dependent Antioxidant Response in Human Epithelial Colon Cells

Journal: Molecules (Basel, Switzerland)

doi: 10.3390/molecules15053338

Upregulation of Nrf2 and HO-1 protein levels in normal human fetal epithelial colon cells exposed to cinnamic aldehyde and ethanolic cinnamon extract. FHC cells were treated with either CA (10 μM), CE (dose CA: 10 μM), or tBHQ (100 μM) for 4 h. Equal loading was assessed by immunodetection of lamin A.
Figure Legend Snippet: Upregulation of Nrf2 and HO-1 protein levels in normal human fetal epithelial colon cells exposed to cinnamic aldehyde and ethanolic cinnamon extract. FHC cells were treated with either CA (10 μM), CE (dose CA: 10 μM), or tBHQ (100 μM) for 4 h. Equal loading was assessed by immunodetection of lamin A.

Techniques Used: Immunodetection

Dose response of Nrf2 transcriptional activation by cinnamic aldehyde and an ethanolic cinnamon extract in MDA-MB231 breast carcinoma and HCT116 colon carcinoma cells. (a–b) MDA-MB-231 cells (panel a) or HCT116 (panel b) were cotransfected with plasmids containing a GST-ARE-firefly luciferase reporter gene and expression plasmids for the Nrf2 and Keap1 proteins. A plasmid encoding renilla luciferase, driven by the herpes simplex virus thymidine kinase promoter, was included in all transfections to normalize transfection efficiency. Twenty-four hours post-transfection, cells were dosed with the indicated concentrations of each compound (tBHQ: 50μM) for 16h. Firefly and renilla luciferase activity was measured and is expressed as relative activity (F/R) compared to untreated control ( p
Figure Legend Snippet: Dose response of Nrf2 transcriptional activation by cinnamic aldehyde and an ethanolic cinnamon extract in MDA-MB231 breast carcinoma and HCT116 colon carcinoma cells. (a–b) MDA-MB-231 cells (panel a) or HCT116 (panel b) were cotransfected with plasmids containing a GST-ARE-firefly luciferase reporter gene and expression plasmids for the Nrf2 and Keap1 proteins. A plasmid encoding renilla luciferase, driven by the herpes simplex virus thymidine kinase promoter, was included in all transfections to normalize transfection efficiency. Twenty-four hours post-transfection, cells were dosed with the indicated concentrations of each compound (tBHQ: 50μM) for 16h. Firefly and renilla luciferase activity was measured and is expressed as relative activity (F/R) compared to untreated control ( p

Techniques Used: Activation Assay, Multiple Displacement Amplification, Luciferase, Expressing, Plasmid Preparation, Transfection, Activity Assay

Upregulation of Nrf2 protein levels in cultured human colon cancer cells exposed to cinnamic aldehyde and ethanolic cinnamon extract. Cultured human HCT116 ( a ) and HT29 cells ( b ) were treated for 4 h with either CA or CE at the indicated doses normalized for CA content. Treatment with tBHQ (100 μM, 4h) served as a positive control. Equal loading was assessed by immunodetection of β-actin.
Figure Legend Snippet: Upregulation of Nrf2 protein levels in cultured human colon cancer cells exposed to cinnamic aldehyde and ethanolic cinnamon extract. Cultured human HCT116 ( a ) and HT29 cells ( b ) were treated for 4 h with either CA or CE at the indicated doses normalized for CA content. Treatment with tBHQ (100 μM, 4h) served as a positive control. Equal loading was assessed by immunodetection of β-actin.

Techniques Used: Cell Culture, Positive Control, Immunodetection

24) Product Images from "Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells"

Article Title: Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.0277

PI activates IκB/NF-κB pathway (A) NCI-H157 and A549 cells were treated with PS-341 (50 nM) or MG132 (20 μM) for the indicated times. Total cellular extracts were subjected to Western blot analysis for IκBα and GAPDH. (B) NCI-H157 cells were treated with PS-341 or MG132 for the indicated times. Nuclear proteins were extracted and subjected to Western blot analysis for p65 and Lamin A/C. TNF-α (10 ng/mL, 60 min) was used as a positive control. (C) Both cell lines were stimulated with PS-341 or MG132 for 0, 6, 8, 12, or 24 h. Total RNA was isolated and quantitative real-time PCR for COX-2 and GAPDH was performed. Data represent the mean ± SD of triplicate experiments. ** P
Figure Legend Snippet: PI activates IκB/NF-κB pathway (A) NCI-H157 and A549 cells were treated with PS-341 (50 nM) or MG132 (20 μM) for the indicated times. Total cellular extracts were subjected to Western blot analysis for IκBα and GAPDH. (B) NCI-H157 cells were treated with PS-341 or MG132 for the indicated times. Nuclear proteins were extracted and subjected to Western blot analysis for p65 and Lamin A/C. TNF-α (10 ng/mL, 60 min) was used as a positive control. (C) Both cell lines were stimulated with PS-341 or MG132 for 0, 6, 8, 12, or 24 h. Total RNA was isolated and quantitative real-time PCR for COX-2 and GAPDH was performed. Data represent the mean ± SD of triplicate experiments. ** P

Techniques Used: Western Blot, Positive Control, Isolation, Real-time Polymerase Chain Reaction

25) Product Images from "Oxidative stress and cell damage in a model of precancerous lesions and advanced hepatocellular carcinoma in rats"

Article Title: Oxidative stress and cell damage in a model of precancerous lesions and advanced hepatocellular carcinoma in rats

Journal: Toxicology Reports

doi: 10.1016/j.toxrep.2014.11.015

Western blot analysis of NQO1, Keap1, Nrf2, HP73 and HP72. Protein from liver extracts was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. (A) Representative images. (B) Densitometric quantification. Values are expressed as means ± SD ( n = 5). a p
Figure Legend Snippet: Western blot analysis of NQO1, Keap1, Nrf2, HP73 and HP72. Protein from liver extracts was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. (A) Representative images. (B) Densitometric quantification. Values are expressed as means ± SD ( n = 5). a p

Techniques Used: Western Blot, Polyacrylamide Gel Electrophoresis

26) Product Images from "Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells"

Article Title: Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.0277

Macroautophagy mediates PI-induced IκBα degradation (A, B) NCI-H157 cells were transiently transfected with control shRNAs, Lamp2 shRNAs, control siRNAs, or LC3B siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) or MG132 (20 μM) for 8 h. Total cellular extracts were subjected to Western blot analysis for IκBα, Lamp2a, Lamp2, LC3B, and GAPDH. Results are representative of three independent experiments.
Figure Legend Snippet: Macroautophagy mediates PI-induced IκBα degradation (A, B) NCI-H157 cells were transiently transfected with control shRNAs, Lamp2 shRNAs, control siRNAs, or LC3B siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) or MG132 (20 μM) for 8 h. Total cellular extracts were subjected to Western blot analysis for IκBα, Lamp2a, Lamp2, LC3B, and GAPDH. Results are representative of three independent experiments.

Techniques Used: Transfection, Western Blot

27) Product Images from "Oxidative stress and cell damage in a model of precancerous lesions and advanced hepatocellular carcinoma in rats"

Article Title: Oxidative stress and cell damage in a model of precancerous lesions and advanced hepatocellular carcinoma in rats

Journal: Toxicology Reports

doi: 10.1016/j.toxrep.2014.11.015

Western blot analysis of TGF-1β, eNOS, and iNOS. Protein from liver extracts was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. (A) Representative images. (B) Densitometric quantification. Values are expressed as means ± SD ( n = 5). a p
Figure Legend Snippet: Western blot analysis of TGF-1β, eNOS, and iNOS. Protein from liver extracts was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. (A) Representative images. (B) Densitometric quantification. Values are expressed as means ± SD ( n = 5). a p

Techniques Used: Western Blot, Polyacrylamide Gel Electrophoresis

28) Product Images from "A specific expression profile of LC3B and p62 is associated with nonresponse to neoadjuvant chemotherapy in esophageal adenocarcinomas"

Article Title: A specific expression profile of LC3B and p62 is associated with nonresponse to neoadjuvant chemotherapy in esophageal adenocarcinomas

Journal: PLoS ONE

doi: 10.1371/journal.pone.0197610

Examples of immunohistochemical stainings. (a) High LC3B dot like staining (score 2). (b) Low LC3B dot like staining (score 1), note a small nerve serving as internal positive control. (c) High p62 cytoplasmic staining (score 3), while negative nuclear staining. (d) Low p62 cytoplasmic/dot-like staining (scores 0), positive nuclear staining. (e) High cytoplasmic (score 2) and low dot-like (score 1) p62 staining. (f) Low cytoplasmic (score 1) and high dot like (score 2) p62 staining. 40x magnification for all images. Error bars indicate 20µm.
Figure Legend Snippet: Examples of immunohistochemical stainings. (a) High LC3B dot like staining (score 2). (b) Low LC3B dot like staining (score 1), note a small nerve serving as internal positive control. (c) High p62 cytoplasmic staining (score 3), while negative nuclear staining. (d) Low p62 cytoplasmic/dot-like staining (scores 0), positive nuclear staining. (e) High cytoplasmic (score 2) and low dot-like (score 1) p62 staining. (f) Low cytoplasmic (score 1) and high dot like (score 2) p62 staining. 40x magnification for all images. Error bars indicate 20µm.

Techniques Used: Immunohistochemistry, Staining, Positive Control

Cytoplasmic expression of p62 results in decreased responsiveness of EAC cells to paclitaxel. (a) OE19 p62 knockdown cells were transiently transfected with either a cytoplasmic or nuclear GFP-tagged p62 expression plasmid. GFP (GFP-p62 fusion proteins) and nuclear DAPI staining as analyzed by confocal microscopy are shown. (b) Annexin V/DAPI fluorescence-activated cell sorting (FACS) analysis of OE19 cells expressing cytoplasmic or nuclear p62 after 48 h of paclitaxel treatment. Bars represent four experimental replicates.
Figure Legend Snippet: Cytoplasmic expression of p62 results in decreased responsiveness of EAC cells to paclitaxel. (a) OE19 p62 knockdown cells were transiently transfected with either a cytoplasmic or nuclear GFP-tagged p62 expression plasmid. GFP (GFP-p62 fusion proteins) and nuclear DAPI staining as analyzed by confocal microscopy are shown. (b) Annexin V/DAPI fluorescence-activated cell sorting (FACS) analysis of OE19 cells expressing cytoplasmic or nuclear p62 after 48 h of paclitaxel treatment. Bars represent four experimental replicates.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Fluorescence, FACS

Kaplan-Meier survival curves for autophagy markers in post-treatment tumor tissue of a neo-adjuvant EAC cohort. (A) LC3B dot-like staining patterns, (B) p62 dot-like staining patterns (C) groupings of LC3B dot-like/p62 dot-like-cytoplasmic expression: Low LC3B/low p62 (LL), low LC3B/high p62 (LH), high LC3B/low p62 (HL) and high LC3B/high p62 (HH); and (D) LC3B dot-like/p62 dot-like-cytoplasmic expression LH versus remainder of all other cases. For each curve the p-value is displayed on the bottom right-hand corner.
Figure Legend Snippet: Kaplan-Meier survival curves for autophagy markers in post-treatment tumor tissue of a neo-adjuvant EAC cohort. (A) LC3B dot-like staining patterns, (B) p62 dot-like staining patterns (C) groupings of LC3B dot-like/p62 dot-like-cytoplasmic expression: Low LC3B/low p62 (LL), low LC3B/high p62 (LH), high LC3B/low p62 (HL) and high LC3B/high p62 (HH); and (D) LC3B dot-like/p62 dot-like-cytoplasmic expression LH versus remainder of all other cases. For each curve the p-value is displayed on the bottom right-hand corner.

Techniques Used: Staining, Expressing

29) Product Images from "A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells"

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

Journal: Autophagy

doi: 10.1080/15548627.2015.1052928

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

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

30) Product Images from "Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels"

Article Title: Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels

Journal: Scientific Reports

doi: 10.1038/s41598-018-26049-5

Effect of plasma on chicken sperm mRNA and protein expression. Semen of 60-week-old cocks was exposed to varying plasma potentials for 20 s. Relative mRNA levels of the following genes were measured: ( a ) NOX4 , NRF2 , and KEAP1 ; ( b ) SOD , CAT , and GPx ; ( c ) PRDX1 , PRDX 3, PRDX4 , and PRDX6 ; ( d ) ATP5A1 , ATP5B , ATP5C1 , ATP5F1 , ATP5G1 , ATP5G3 , ATP5H , ATP5I , ATP5J , ATP5J 2, ATP5L , and ATP5S ; and ( e ) AMPKα2 , AMPKβ2 , AMPKγ3 , and mTOR . ( f . The grouping of gels/blots cropped from different gels. All blots were visualized with 5 min exposure time. Relative protein levels of ( g ) NRF2, KEAP1, PRDX4, ( h ) ATP5A, ( i ) p-AMPKα/AMPKα, and ( j ) p-mTOR/mTOR. Values are expressed as the mean ± standard error (n = 3) of three replicates; n represents an individual cock. * p
Figure Legend Snippet: Effect of plasma on chicken sperm mRNA and protein expression. Semen of 60-week-old cocks was exposed to varying plasma potentials for 20 s. Relative mRNA levels of the following genes were measured: ( a ) NOX4 , NRF2 , and KEAP1 ; ( b ) SOD , CAT , and GPx ; ( c ) PRDX1 , PRDX 3, PRDX4 , and PRDX6 ; ( d ) ATP5A1 , ATP5B , ATP5C1 , ATP5F1 , ATP5G1 , ATP5G3 , ATP5H , ATP5I , ATP5J , ATP5J 2, ATP5L , and ATP5S ; and ( e ) AMPKα2 , AMPKβ2 , AMPKγ3 , and mTOR . ( f . The grouping of gels/blots cropped from different gels. All blots were visualized with 5 min exposure time. Relative protein levels of ( g ) NRF2, KEAP1, PRDX4, ( h ) ATP5A, ( i ) p-AMPKα/AMPKα, and ( j ) p-mTOR/mTOR. Values are expressed as the mean ± standard error (n = 3) of three replicates; n represents an individual cock. * p

Techniques Used: Expressing

31) Product Images from "Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells"

Article Title: Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.0277

Macroautophagy mediates PI-induced IκBα degradation (A, B) NCI-H157 cells were transiently transfected with control shRNAs, Lamp2 shRNAs, control siRNAs, or LC3B siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) or MG132 (20 μM) for 8 h. Total cellular extracts were subjected to Western blot analysis for IκBα, Lamp2a, Lamp2, LC3B, and GAPDH. Results are representative of three independent experiments.
Figure Legend Snippet: Macroautophagy mediates PI-induced IκBα degradation (A, B) NCI-H157 cells were transiently transfected with control shRNAs, Lamp2 shRNAs, control siRNAs, or LC3B siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) or MG132 (20 μM) for 8 h. Total cellular extracts were subjected to Western blot analysis for IκBα, Lamp2a, Lamp2, LC3B, and GAPDH. Results are representative of three independent experiments.

Techniques Used: Transfection, Western Blot

32) Product Images from "Schisandrol B protects against acetaminophen-induced acute hepatotoxicity in mice via activation of the NRF2/ARE signaling pathway"

Article Title: Schisandrol B protects against acetaminophen-induced acute hepatotoxicity in mice via activation of the NRF2/ARE signaling pathway

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2015.120

The effects of SolB on activation and expression of NRF2 in vivo . (A) qRT-PCR analysis was performed to measure the gene expression of Nrf2, and the data are expressed as the mean±SEM ( n =6). (B–C) Western blotting was used to measure nuclear NRF2 protein expression. Specific band intensities were quantified and normalized to histone H3 and expressed as the mean±SEM ( n =3). (D–E) Western blotting was used to measure cytoplasmic KEAP1 protein expression. Specific band intensities were quantified and normalized to GAPDH and expressed as the mean±SEM ( n =3). b P
Figure Legend Snippet: The effects of SolB on activation and expression of NRF2 in vivo . (A) qRT-PCR analysis was performed to measure the gene expression of Nrf2, and the data are expressed as the mean±SEM ( n =6). (B–C) Western blotting was used to measure nuclear NRF2 protein expression. Specific band intensities were quantified and normalized to histone H3 and expressed as the mean±SEM ( n =3). (D–E) Western blotting was used to measure cytoplasmic KEAP1 protein expression. Specific band intensities were quantified and normalized to GAPDH and expressed as the mean±SEM ( n =3). b P

Techniques Used: Activation Assay, Expressing, In Vivo, Quantitative RT-PCR, Western Blot

Role of NRF2 signaling and its downstream proteins in SolB-mediated protection against APAP-induced liver toxicity. (A) Western blotting was used to measure the protein expression of GSR, GSS, GCLC, GCLM, GST-α, GST-μ, GST-π, NQO1, HO-1, MRP2, MRP3 and MRP4. (B–M) Specific band intensities were quantified and normalized to GAPDH. The data are expressed as the mean±SEM ( n =3). b P
Figure Legend Snippet: Role of NRF2 signaling and its downstream proteins in SolB-mediated protection against APAP-induced liver toxicity. (A) Western blotting was used to measure the protein expression of GSR, GSS, GCLC, GCLM, GST-α, GST-μ, GST-π, NQO1, HO-1, MRP2, MRP3 and MRP4. (B–M) Specific band intensities were quantified and normalized to GAPDH. The data are expressed as the mean±SEM ( n =3). b P

Techniques Used: Western Blot, Expressing

Effects of SolB on cell viability and NRF2 activation. (A) An MTT assay was used to measure HepG2 cell viability after treatment with SolB at 2.5 to 160 μmol/L. (B) A luciferase reporter assay was used to measure the effect of SolB on NRF2 activation. The HepG2 cells were transiently transfected with plasmids as described in the Materials and methods section, and 6 h later the cells were treated with various concentrations of SolB (2.5-20 μmol/L) or the positive agonist SFN (10 μmol/L) for 24 h. The data are expressed as the mean±SEM ( n =5). c P
Figure Legend Snippet: Effects of SolB on cell viability and NRF2 activation. (A) An MTT assay was used to measure HepG2 cell viability after treatment with SolB at 2.5 to 160 μmol/L. (B) A luciferase reporter assay was used to measure the effect of SolB on NRF2 activation. The HepG2 cells were transiently transfected with plasmids as described in the Materials and methods section, and 6 h later the cells were treated with various concentrations of SolB (2.5-20 μmol/L) or the positive agonist SFN (10 μmol/L) for 24 h. The data are expressed as the mean±SEM ( n =5). c P

Techniques Used: Activation Assay, MTT Assay, Luciferase, Reporter Assay, Transfection

33) 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

Feedback activation of the Keap1-Nrf2 pathway serves as a physiological adaptation to impaired proteasome function. A , total lysates, detergent-soluble ( Sol. ) and -insoluble ( Insol. ) fractions, and nuclear fractions from livers of the indicated genotypes
Figure Legend Snippet: Feedback activation of the Keap1-Nrf2 pathway serves as a physiological adaptation to impaired proteasome function. A , total lysates, detergent-soluble ( Sol. ) and -insoluble ( Insol. ) fractions, and nuclear fractions from livers of the indicated genotypes

Techniques Used: Activation Assay

Role of p62 in formation of aggregate-containing structures and Nrf2 activation in Rpt2 f/f ;Alb hepatocytes. A , total lysates, detergent-soluble ( Sol. ) and -insoluble ( Insol. ) fractions, and nuclear fractions from livers of the indicated genotypes were
Figure Legend Snippet: Role of p62 in formation of aggregate-containing structures and Nrf2 activation in Rpt2 f/f ;Alb hepatocytes. A , total lysates, detergent-soluble ( Sol. ) and -insoluble ( Insol. ) fractions, and nuclear fractions from livers of the indicated genotypes were

Techniques Used: Activation Assay

34) Product Images from "Protective Effects of Berberine on Renal Injury in Streptozotocin (STZ)-Induced Diabetic Mice"

Article Title: Protective Effects of Berberine on Renal Injury in Streptozotocin (STZ)-Induced Diabetic Mice

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17081327

BBR enhanced activation of Nrf2/HO-1 signaling in diabetic kidneys. Representative western blots demonstrated that BBR treatment increased protein expression of Nrf2, NQO1 and HO-1 in the kidneys of diabetic mice ( n = 8). Each value represents mean ± SEM. All experiments were performed three times with virtually identical results. β-actin was used as loading control. (** p
Figure Legend Snippet: BBR enhanced activation of Nrf2/HO-1 signaling in diabetic kidneys. Representative western blots demonstrated that BBR treatment increased protein expression of Nrf2, NQO1 and HO-1 in the kidneys of diabetic mice ( n = 8). Each value represents mean ± SEM. All experiments were performed three times with virtually identical results. β-actin was used as loading control. (** p

Techniques Used: Activation Assay, Western Blot, Expressing, Mouse Assay

Antifibrotic effects of BBR were associated with the inhibition of EMT in diabetic kidneys. Representative western blots of α-SMA and collagen-I in the kidneys of control mice, diabetic mice or BBR-treated diabetic mice. All experiments were performed three times with virtually identical results. β-actin was used as loading control. (** p
Figure Legend Snippet: Antifibrotic effects of BBR were associated with the inhibition of EMT in diabetic kidneys. Representative western blots of α-SMA and collagen-I in the kidneys of control mice, diabetic mice or BBR-treated diabetic mice. All experiments were performed three times with virtually identical results. β-actin was used as loading control. (** p

Techniques Used: Inhibition, Western Blot, Mouse Assay

Influence of BBR on HG-induced Nrf2/HO-1 signaling in NRK-52E cells . The NRK-52E cells were treated with 30 mM HG for 48 h, in the presence or absence of BBR. The expression NQO1, HO-1 and Nrf2 was analyzed using western blot ( n = 6). ( a – e ) The results were representatives of three independent experiments. β-actin was used as loading control. (** p
Figure Legend Snippet: Influence of BBR on HG-induced Nrf2/HO-1 signaling in NRK-52E cells . The NRK-52E cells were treated with 30 mM HG for 48 h, in the presence or absence of BBR. The expression NQO1, HO-1 and Nrf2 was analyzed using western blot ( n = 6). ( a – e ) The results were representatives of three independent experiments. β-actin was used as loading control. (** p

Techniques Used: Expressing, Western Blot

35) 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

36) Product Images from "A novel cystine based antioxidant attenuates oxidative stress and hepatic steatosis in diet-induced obese mice"

Article Title: A novel cystine based antioxidant attenuates oxidative stress and hepatic steatosis in diet-induced obese mice

Journal: Experimental and molecular pathology

doi: 10.1016/j.yexmp.2011.04.009

F1 treatment prevents activation of JNK and p38 MAPK, perturbation of BAX/BCL-2 ratio, and caspase activation in livers in mice fed with HFD. A: EIA revels significantly (P
Figure Legend Snippet: F1 treatment prevents activation of JNK and p38 MAPK, perturbation of BAX/BCL-2 ratio, and caspase activation in livers in mice fed with HFD. A: EIA revels significantly (P

Techniques Used: Activation Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

37) Product Images from "Effect of p62 on tau hyperphosphorylation in a rat model of Alzheimer's disease ☆"

Article Title: Effect of p62 on tau hyperphosphorylation in a rat model of Alzheimer's disease ☆

Journal: Neural Regeneration Research

doi: 10.3969/j.issn.1673-5374.2012.17.004

Changes in Keap1 and Nrf2 expression in the rat cerebral cortex and hippocampus. (A, B) Immunohistochemical and quantitative analysis for the expression of Keap1 in the cerebral cortex of Alzheimer's disease (AD) model rats. The expression of Keap1 in AD model rats was increased compared with the control (CON) group. (C, D) Immunohistochemical and quantitative analysis for the expression of Nrf2 in the cerebral cortex of AD model rats. The expression of Nrf2 in AD model rats increased compared with control rats. (E, F) Immunohistochemical and quantitative analysis for the expression of Keap1 in the hippocampus of AD model rats. The expression of Keap1 in AD model rats increased compared with control rats. (G, H) Immunohistochemical and quantitative analysis for the expression of Nrf2 in the hippocampus of AD model rats. The expression of Nrf2 in AD model rats remained unchanged compared with control rats. Scale bars: 20 μm. a P
Figure Legend Snippet: Changes in Keap1 and Nrf2 expression in the rat cerebral cortex and hippocampus. (A, B) Immunohistochemical and quantitative analysis for the expression of Keap1 in the cerebral cortex of Alzheimer's disease (AD) model rats. The expression of Keap1 in AD model rats was increased compared with the control (CON) group. (C, D) Immunohistochemical and quantitative analysis for the expression of Nrf2 in the cerebral cortex of AD model rats. The expression of Nrf2 in AD model rats increased compared with control rats. (E, F) Immunohistochemical and quantitative analysis for the expression of Keap1 in the hippocampus of AD model rats. The expression of Keap1 in AD model rats increased compared with control rats. (G, H) Immunohistochemical and quantitative analysis for the expression of Nrf2 in the hippocampus of AD model rats. The expression of Nrf2 in AD model rats remained unchanged compared with control rats. Scale bars: 20 μm. a P

Techniques Used: Expressing, Immunohistochemistry

38) Product Images from "Attenuation of oxygen fluctuation-induced endoplasmic reticulum stress in human lens epithelial cells"

Article Title: Attenuation of oxygen fluctuation-induced endoplasmic reticulum stress in human lens epithelial cells

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2015.2725

Graphical representation of mRNA quantification of antioxidants Nrf2 and Keap1 in hLECs cultured for 24 h with 0, 1, 4 and 20% oxygen. *P
Figure Legend Snippet: Graphical representation of mRNA quantification of antioxidants Nrf2 and Keap1 in hLECs cultured for 24 h with 0, 1, 4 and 20% oxygen. *P

Techniques Used: Cell Culture

39) Product Images from "Negative Regulation of the Keap1-Nrf2 Pathway by a p62/Sqstm1 Splicing Variant"

Article Title: Negative Regulation of the Keap1-Nrf2 Pathway by a p62/Sqstm1 Splicing Variant

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00642-17

The p62 variant negatively regulates the p62-Keap1-Nrf2 axis in mouse hepatocytes. (A) Schematic diagram of the genome structures of wild-type p62 and p62-GFP knock-in alleles. Coding exons, numbered in accordance with the initiation site (exon 1), are depicted by white boxes. E2-cDNA–GFP–pA indicates the p62 cDNA fragment (positions 302 to 1326) fused with GFP cDNA and simian virus 40 (SV40) poly(A). mRNAs and proteins produced from each allele are shown. (B) Immunoblot analysis. Primary hepatocytes prepared from both wild-type and p62-GFP knock-in mice were challenged with 10 μM sodium arsenite [As(III)] for 12 h. After removal of As(III), cells were cultured in regular medium for the indicated times. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data show the results of two independent experiments. Numerical values indicate the results of quantitative densitometric analyses of Nqo1 normalized against the levels in nontreated cells. An asterisk indicates a nonspecific band. (C) Real-time PCR. Relative mRNA levels of Nrf2 targets in hepatocytes prepared as described for panel B are shown. Values were normalized against the corresponding mRNA levels in nontreated wild-type or p62-GFP knock-in hepatocytes. The experiments were performed two times.
Figure Legend Snippet: The p62 variant negatively regulates the p62-Keap1-Nrf2 axis in mouse hepatocytes. (A) Schematic diagram of the genome structures of wild-type p62 and p62-GFP knock-in alleles. Coding exons, numbered in accordance with the initiation site (exon 1), are depicted by white boxes. E2-cDNA–GFP–pA indicates the p62 cDNA fragment (positions 302 to 1326) fused with GFP cDNA and simian virus 40 (SV40) poly(A). mRNAs and proteins produced from each allele are shown. (B) Immunoblot analysis. Primary hepatocytes prepared from both wild-type and p62-GFP knock-in mice were challenged with 10 μM sodium arsenite [As(III)] for 12 h. After removal of As(III), cells were cultured in regular medium for the indicated times. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data show the results of two independent experiments. Numerical values indicate the results of quantitative densitometric analyses of Nqo1 normalized against the levels in nontreated cells. An asterisk indicates a nonspecific band. (C) Real-time PCR. Relative mRNA levels of Nrf2 targets in hepatocytes prepared as described for panel B are shown. Values were normalized against the corresponding mRNA levels in nontreated wild-type or p62-GFP knock-in hepatocytes. The experiments were performed two times.

Techniques Used: Variant Assay, Knock-In, Produced, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction

The p62 variant increases the amount of Keap1 and represses Nrf2 activity. (A) Real-time PCR. Relative mRNA levels of Nrf2 targets in primary mouse hepatocytes expressing full-length p62 or variant p62 at the indicated multiplicities of infection (MOIs) are shown. Values were normalized to the amount of each mRNA in the nontreated wild-type hepatocytes. The experiments were performed three times. Data are means ± standard errors of the means (SEM). *, P
Figure Legend Snippet: The p62 variant increases the amount of Keap1 and represses Nrf2 activity. (A) Real-time PCR. Relative mRNA levels of Nrf2 targets in primary mouse hepatocytes expressing full-length p62 or variant p62 at the indicated multiplicities of infection (MOIs) are shown. Values were normalized to the amount of each mRNA in the nontreated wild-type hepatocytes. The experiments were performed three times. Data are means ± standard errors of the means (SEM). *, P

Techniques Used: Variant Assay, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Infection

40) Product Images from "Reduced mammalian target of rapamycin activity facilitates mitochondrial retrograde signaling and increases life span in normal human fibroblasts"

Article Title: Reduced mammalian target of rapamycin activity facilitates mitochondrial retrograde signaling and increases life span in normal human fibroblasts

Journal: Aging cell

doi: 10.1111/acel.12122

Inhibition of mammalian target of rapamycin (mTOR) increases the levels of proteins associated with mitochondrial biogenesis and activation of NFE2L2. (A) Representative Western blots of the steady-state levels of PGC-1α, TFAm, NFE2L2, NRF1, and
Figure Legend Snippet: Inhibition of mammalian target of rapamycin (mTOR) increases the levels of proteins associated with mitochondrial biogenesis and activation of NFE2L2. (A) Representative Western blots of the steady-state levels of PGC-1α, TFAm, NFE2L2, NRF1, and

Techniques Used: Inhibition, Activation Assay, Western Blot, Pyrolysis Gas Chromatography

41) Product Images from "MiR-28 regulates Nrf2 expression through a Keap1-independent mechanism"

Article Title: MiR-28 regulates Nrf2 expression through a Keap1-independent mechanism

Journal: Breast cancer research and treatment

doi: 10.1007/s10549-011-1604-1

miR-28 regulation of Nrf2 expression is Keap1 independent. a MCF-7 cells were co-transfected with Keap1-FLAG and pri-miR-28 or vehicle-control. Western blotting was used to detect Keap1 expression with anti-FLAG antibody. β -actin was used as a loading control. b Keap1-FLAG and Nrf2-myc were co-transfected into HEK293T cells with pri-miR-28 or vehicle control. Co-immunoprecipitation was performed to examine Nrf2-Keap1 interaction status. The anti-myc antibody was used to pull down antigens and the mouse IgG was used as negative control; anti-FLAG or anti-myc antibody was used for Western blotting. A representative experiment was shown. Normalized protein expression in input was analyzed using UN-SCAN-IT program
Figure Legend Snippet: miR-28 regulation of Nrf2 expression is Keap1 independent. a MCF-7 cells were co-transfected with Keap1-FLAG and pri-miR-28 or vehicle-control. Western blotting was used to detect Keap1 expression with anti-FLAG antibody. β -actin was used as a loading control. b Keap1-FLAG and Nrf2-myc were co-transfected into HEK293T cells with pri-miR-28 or vehicle control. Co-immunoprecipitation was performed to examine Nrf2-Keap1 interaction status. The anti-myc antibody was used to pull down antigens and the mouse IgG was used as negative control; anti-FLAG or anti-myc antibody was used for Western blotting. A representative experiment was shown. Normalized protein expression in input was analyzed using UN-SCAN-IT program

Techniques Used: Expressing, Transfection, Western Blot, Immunoprecipitation, Negative Control

42) Product Images from "Negative Regulation of the Keap1-Nrf2 Pathway by a p62/Sqstm1 Splicing Variant"

Article Title: Negative Regulation of the Keap1-Nrf2 Pathway by a p62/Sqstm1 Splicing Variant

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00642-17

The p62 variant negatively regulates the p62-Keap1-Nrf2 axis in mouse hepatocytes. (A) Schematic diagram of the genome structures of wild-type p62 and p62-GFP knock-in alleles. Coding exons, numbered in accordance with the initiation site (exon 1), are depicted by white boxes. E2-cDNA–GFP–pA indicates the p62 cDNA fragment (positions 302 to 1326) fused with GFP cDNA and simian virus 40 (SV40) poly(A). mRNAs and proteins produced from each allele are shown. (B) Immunoblot analysis. Primary hepatocytes prepared from both wild-type and p62-GFP knock-in mice were challenged with 10 μM sodium arsenite [As(III)] for 12 h. After removal of As(III), cells were cultured in regular medium for the indicated times. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data show the results of two independent experiments. Numerical values indicate the results of quantitative densitometric analyses of Nqo1 normalized against the levels in nontreated cells. An asterisk indicates a nonspecific band. (C) Real-time PCR. Relative mRNA levels of Nrf2 targets in hepatocytes prepared as described for panel B are shown. Values were normalized against the corresponding mRNA levels in nontreated wild-type or p62-GFP knock-in hepatocytes. The experiments were performed two times.
Figure Legend Snippet: The p62 variant negatively regulates the p62-Keap1-Nrf2 axis in mouse hepatocytes. (A) Schematic diagram of the genome structures of wild-type p62 and p62-GFP knock-in alleles. Coding exons, numbered in accordance with the initiation site (exon 1), are depicted by white boxes. E2-cDNA–GFP–pA indicates the p62 cDNA fragment (positions 302 to 1326) fused with GFP cDNA and simian virus 40 (SV40) poly(A). mRNAs and proteins produced from each allele are shown. (B) Immunoblot analysis. Primary hepatocytes prepared from both wild-type and p62-GFP knock-in mice were challenged with 10 μM sodium arsenite [As(III)] for 12 h. After removal of As(III), cells were cultured in regular medium for the indicated times. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data show the results of two independent experiments. Numerical values indicate the results of quantitative densitometric analyses of Nqo1 normalized against the levels in nontreated cells. An asterisk indicates a nonspecific band. (C) Real-time PCR. Relative mRNA levels of Nrf2 targets in hepatocytes prepared as described for panel B are shown. Values were normalized against the corresponding mRNA levels in nontreated wild-type or p62-GFP knock-in hepatocytes. The experiments were performed two times.

Techniques Used: Variant Assay, Knock-In, Produced, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction

The p62 variant increases the amount of Keap1 and represses Nrf2 activity. (A) Real-time PCR. Relative mRNA levels of Nrf2 targets in primary mouse hepatocytes expressing full-length p62 or variant p62 at the indicated multiplicities of infection (MOIs) are shown. Values were normalized to the amount of each mRNA in the nontreated wild-type hepatocytes. The experiments were performed three times. Data are means ± standard errors of the means (SEM). *, P
Figure Legend Snippet: The p62 variant increases the amount of Keap1 and represses Nrf2 activity. (A) Real-time PCR. Relative mRNA levels of Nrf2 targets in primary mouse hepatocytes expressing full-length p62 or variant p62 at the indicated multiplicities of infection (MOIs) are shown. Values were normalized to the amount of each mRNA in the nontreated wild-type hepatocytes. The experiments were performed three times. Data are means ± standard errors of the means (SEM). *, P

Techniques Used: Variant Assay, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Infection

43) Product Images from "Protective Effects of Berberine on Renal Injury in Streptozotocin (STZ)-Induced Diabetic Mice"

Article Title: Protective Effects of Berberine on Renal Injury in Streptozotocin (STZ)-Induced Diabetic Mice

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17081327

siRNA knockdown of Nrf2 abrogates BBR-induced NQO1 and HO-1 expression. ( a ) NRK-52E cells were transfected with Nrf2-siRNA and western blot analysis was performed with an antibody against Nrf2 was performed at various time-points following transfection (24, 48 and 72 h). Relative Nrf2 expression levels were calculated and normalized to the loading control. Corresponding protein levels were assessed using densitometry and expressed in relative intensities. All results were obtained from three independent experiments. Values are expressed as the mean ± SEM ( n = 6; ** p
Figure Legend Snippet: siRNA knockdown of Nrf2 abrogates BBR-induced NQO1 and HO-1 expression. ( a ) NRK-52E cells were transfected with Nrf2-siRNA and western blot analysis was performed with an antibody against Nrf2 was performed at various time-points following transfection (24, 48 and 72 h). Relative Nrf2 expression levels were calculated and normalized to the loading control. Corresponding protein levels were assessed using densitometry and expressed in relative intensities. All results were obtained from three independent experiments. Values are expressed as the mean ± SEM ( n = 6; ** p

Techniques Used: Expressing, Transfection, Western Blot

BBR enhanced activation of Nrf2/HO-1 signaling in diabetic kidneys. Representative western blots demonstrated that BBR treatment increased protein expression of Nrf2, NQO1 and HO-1 in the kidneys of diabetic mice ( n = 8). Each value represents mean ± SEM. All experiments were performed three times with virtually identical results. β-actin was used as loading control. (** p
Figure Legend Snippet: BBR enhanced activation of Nrf2/HO-1 signaling in diabetic kidneys. Representative western blots demonstrated that BBR treatment increased protein expression of Nrf2, NQO1 and HO-1 in the kidneys of diabetic mice ( n = 8). Each value represents mean ± SEM. All experiments were performed three times with virtually identical results. β-actin was used as loading control. (** p

Techniques Used: Activation Assay, Western Blot, Expressing, Mouse Assay

Influence of BBR on HG-induced Nrf2/HO-1 signaling in NRK-52E cells . The NRK-52E cells were treated with 30 mM HG for 48 h, in the presence or absence of BBR. The expression NQO1, HO-1 and Nrf2 was analyzed using western blot ( n = 6). ( a – e ) The results were representatives of three independent experiments. β-actin was used as loading control. (** p
Figure Legend Snippet: Influence of BBR on HG-induced Nrf2/HO-1 signaling in NRK-52E cells . The NRK-52E cells were treated with 30 mM HG for 48 h, in the presence or absence of BBR. The expression NQO1, HO-1 and Nrf2 was analyzed using western blot ( n = 6). ( a – e ) The results were representatives of three independent experiments. β-actin was used as loading control. (** p

Techniques Used: Expressing, Western Blot

Nrf2 is involved in the inhibitory effect of BBR on the TGF-β/Smad signaling pathway. NRK-52E cells were divided into six groups as mentioned above, corresponding protein levels of Smad2/3, phospho-Smad2/3 and EMT markers were assessed using densitometry and expressed in relative intensities. All results were obtained from three independent experiments. All results were obtained from three independent experiments. Values are expressed as the mean ± SEM ( n = 6) (* p
Figure Legend Snippet: Nrf2 is involved in the inhibitory effect of BBR on the TGF-β/Smad signaling pathway. NRK-52E cells were divided into six groups as mentioned above, corresponding protein levels of Smad2/3, phospho-Smad2/3 and EMT markers were assessed using densitometry and expressed in relative intensities. All results were obtained from three independent experiments. All results were obtained from three independent experiments. Values are expressed as the mean ± SEM ( n = 6) (* p

Techniques Used:

44) Product Images from "Attenuation of oxygen fluctuation-induced endoplasmic reticulum stress in human lens epithelial cells"

Article Title: Attenuation of oxygen fluctuation-induced endoplasmic reticulum stress in human lens epithelial cells

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2015.2725

Western blot analysis of ROS- and apoptosis-related UPR proteins. (A) ROS-related and (B) apoptosis-related UPR proteins in the hLECs cultured for 24 h with 0, 1, 4 and 20% oxygen. ROS, reactive oxygen species; UPR, unfolded protein response; Ero1-L, endoplasmic reticulum oxidoreductin 1-like; PDI, protein disulfide isomerase; ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein.
Figure Legend Snippet: Western blot analysis of ROS- and apoptosis-related UPR proteins. (A) ROS-related and (B) apoptosis-related UPR proteins in the hLECs cultured for 24 h with 0, 1, 4 and 20% oxygen. ROS, reactive oxygen species; UPR, unfolded protein response; Ero1-L, endoplasmic reticulum oxidoreductin 1-like; PDI, protein disulfide isomerase; ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein.

Techniques Used: Western Blot, Cell Culture

45) Product Images from "The Keap1-BTB Protein Is an Adaptor That Bridges Nrf2 to a Cul3-Based E3 Ligase: Oxidative Stress Sensing by a Cul3-Keap1 Ligase"

Article Title: The Keap1-BTB Protein Is an Adaptor That Bridges Nrf2 to a Cul3-Based E3 Ligase: Oxidative Stress Sensing by a Cul3-Keap1 Ligase

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.19.8477-8486.2004

Cul3 interacts with Keap1 through its BTB domain. (a) 293T cells transfected with plasmids encoding Myc-Keap1 and Flag-Cul3 were either left untreated (lanes 1 to 3) or treated with 5 μg/ of tunicamycin per ml for 1 h (lane 4). Flag-Cul3 was precipitated from whole-cell extracts with the M2 monoclonal antibody; Flag-Cul3 (middle panel) and Cul3-associated Keap1 were detected by immunoblot analysis with the 9E10 antibody (top panel). The presence of ectopically expressed Myc-Keap1 was confirmed by immunoblot analysis with the 9E10 antibody (bottom panel). Lane 1 is a control precipitation with an irrelevant antibody. (b) 293T cells were transfected with plasmids encoding Myc-Keap1, Myc-Keap1ΔBTB, or Flag-Cul3. Flag-Cul3 was precipitated from whole-cell lysates with the M2 monoclonal antibody, and Cul3-associated Keap1 was detected by immunoblot with the 9E10 antibody. Expression of ectopically expressed proteins was confirmed via immunoblot analysis. Lane 1 is a control precipitation with a nonspecific antiserum. (c) NIH 3T3 cells proliferating on glass coverslips were transfected with plasmids expressing Myc-Keap1 and Flag-Cul3. Cells were fixed and examined by indirect immunofluorescence for the presence of the Myc and Flag epitopes.
Figure Legend Snippet: Cul3 interacts with Keap1 through its BTB domain. (a) 293T cells transfected with plasmids encoding Myc-Keap1 and Flag-Cul3 were either left untreated (lanes 1 to 3) or treated with 5 μg/ of tunicamycin per ml for 1 h (lane 4). Flag-Cul3 was precipitated from whole-cell extracts with the M2 monoclonal antibody; Flag-Cul3 (middle panel) and Cul3-associated Keap1 were detected by immunoblot analysis with the 9E10 antibody (top panel). The presence of ectopically expressed Myc-Keap1 was confirmed by immunoblot analysis with the 9E10 antibody (bottom panel). Lane 1 is a control precipitation with an irrelevant antibody. (b) 293T cells were transfected with plasmids encoding Myc-Keap1, Myc-Keap1ΔBTB, or Flag-Cul3. Flag-Cul3 was precipitated from whole-cell lysates with the M2 monoclonal antibody, and Cul3-associated Keap1 was detected by immunoblot with the 9E10 antibody. Expression of ectopically expressed proteins was confirmed via immunoblot analysis. Lane 1 is a control precipitation with a nonspecific antiserum. (c) NIH 3T3 cells proliferating on glass coverslips were transfected with plasmids expressing Myc-Keap1 and Flag-Cul3. Cells were fixed and examined by indirect immunofluorescence for the presence of the Myc and Flag epitopes.

Techniques Used: Transfection, Expressing, Immunofluorescence

In vitro reconstitution of Nrf2 ubiquitination. (a) In vitro-transcribed and -translated Nrf2 was mixed with ATP, ubiquitin, Ubc5, and in vitro-transcribed and -translated Keap1 in in vitro ubiquitination assays for 1 h at 30 o C. In lanes 7 to 10, the reactions were carried out for 15, 30, 60, and 90 min, respectively. (b) In vitro-transcribed and -translated Keap1 (lane 1), Keap1ΔBTB (lane 4), Keap1ΔKelch (lane 3), or Calicin (lane 2) was mixed with in vitro-transcribed and -translated Nrf2, ATP, ubiquitin, and Ubc5 in in vitro ubiquitination assays, as in a, for 1 h at 30°C. (c) In vitro-transcribed and -translated Keap1 (lanes 1 to 3), Keap1ΔBTB (lanes 4 to 6), Keap1ΔKelch (lanes 7 to 9), and Calicin (lanes 10 to 12) were mixed with GST-Nrf2 (lanes 3, 6, 9, and 12) or the negative control, GST (lanes 2, 5, 8, and 11) and washed, and proteins were resolved via SDS-PAGE. Lanes 1, 4, 7, and 10 show 10% input. Proteins were visualized via autoradiography.
Figure Legend Snippet: In vitro reconstitution of Nrf2 ubiquitination. (a) In vitro-transcribed and -translated Nrf2 was mixed with ATP, ubiquitin, Ubc5, and in vitro-transcribed and -translated Keap1 in in vitro ubiquitination assays for 1 h at 30 o C. In lanes 7 to 10, the reactions were carried out for 15, 30, 60, and 90 min, respectively. (b) In vitro-transcribed and -translated Keap1 (lane 1), Keap1ΔBTB (lane 4), Keap1ΔKelch (lane 3), or Calicin (lane 2) was mixed with in vitro-transcribed and -translated Nrf2, ATP, ubiquitin, and Ubc5 in in vitro ubiquitination assays, as in a, for 1 h at 30°C. (c) In vitro-transcribed and -translated Keap1 (lanes 1 to 3), Keap1ΔBTB (lanes 4 to 6), Keap1ΔKelch (lanes 7 to 9), and Calicin (lanes 10 to 12) were mixed with GST-Nrf2 (lanes 3, 6, 9, and 12) or the negative control, GST (lanes 2, 5, 8, and 11) and washed, and proteins were resolved via SDS-PAGE. Lanes 1, 4, 7, and 10 show 10% input. Proteins were visualized via autoradiography.

Techniques Used: In Vitro, Negative Control, SDS Page, Autoradiography

46) Product Images from "Decreased DJ-1 Leads to Impaired Nrf2-Regulated Antioxidant Defense and Increased UV-A–Induced Apoptosis in Corneal Endothelial Cells"

Article Title: Decreased DJ-1 Leads to Impaired Nrf2-Regulated Antioxidant Defense and Increased UV-A–Induced Apoptosis in Corneal Endothelial Cells

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.14-14580

The effect of DJ-1 downregulation on Nrf2-associated Cul3 and Keap1 levels. Immunoprecipitation–WB analysis of Cul3/Nrf2 and Keap/Nrf2 levels in non–siRNA-treated, control siRNA-treated, and DJ-1 siRNA-treated HCECi cells. ( A ) Left panel
Figure Legend Snippet: The effect of DJ-1 downregulation on Nrf2-associated Cul3 and Keap1 levels. Immunoprecipitation–WB analysis of Cul3/Nrf2 and Keap/Nrf2 levels in non–siRNA-treated, control siRNA-treated, and DJ-1 siRNA-treated HCECi cells. ( A ) Left panel

Techniques Used: Immunoprecipitation, Western Blot

DJ-1 downregulation decreases Nrf2 nuclear translocation and its target gene expression. Nrf2 subcellular localization. ( A ) Representative Western blot of Nrf2 protein levels in the cytosolic and nuclear fractions of non–siRNA-treated, control
Figure Legend Snippet: DJ-1 downregulation decreases Nrf2 nuclear translocation and its target gene expression. Nrf2 subcellular localization. ( A ) Representative Western blot of Nrf2 protein levels in the cytosolic and nuclear fractions of non–siRNA-treated, control

Techniques Used: Translocation Assay, Expressing, Western Blot

47) Product Images from "Sulforaphane Protects the Liver against CdSe Quantum Dot-Induced Cytotoxicity"

Article Title: Sulforaphane Protects the Liver against CdSe Quantum Dot-Induced Cytotoxicity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0138771

Effect of knockdown TR-1, Keap1 and Nrf2 on cytotoxicity in HHL-5 cells exposed to CdSe QDs. TR-1, Keap1 or Nrf2 were knocked down, respectively. Allstars (AS) were used as a negative control. Cells were incubated with 5 μM SFN or DMSO (0.1%) control for 24 h then exposed to 20 μM CdSe QDs for 24 h. Cell viability was measured by MTT assay, and data shown as means ± SD (n = 6). Significant levels (*P
Figure Legend Snippet: Effect of knockdown TR-1, Keap1 and Nrf2 on cytotoxicity in HHL-5 cells exposed to CdSe QDs. TR-1, Keap1 or Nrf2 were knocked down, respectively. Allstars (AS) were used as a negative control. Cells were incubated with 5 μM SFN or DMSO (0.1%) control for 24 h then exposed to 20 μM CdSe QDs for 24 h. Cell viability was measured by MTT assay, and data shown as means ± SD (n = 6). Significant levels (*P

Techniques Used: Negative Control, Incubation, MTT Assay

48) Product Images from "Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling"

Article Title: Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling

Journal: The FASEB Journal

doi: 10.1096/fj.201601155R

Proposed mechanism for the effect of FPS-ZM1 in COPD. Exposure to CSE and release of elastase by injured cells during smoking increase ligands, such as S100 calgranulins and HMGB1. The ligands bind to and activate response by RAGE. The engagement of RAGE is linked to the DAMP signaling pathway, notably NF-κB and MAPKs, which overlap considerably with the Nrf2 signaling pathway for RAGE-mediated emphysematous response to stress stimuli in the lung (airspace enlargement, sustained inflammation, excessive ROS/RNS, immune cell infiltration, and epithelial cell death). When mice exposed to PPE or epithelial cells exposed to CSE are treated with FPS-ZM1, RAGE-mediated emphysema and DAMP signaling pathway recover. However, FPS-ZM1 treatment of Nrf2-deficient mice after elastase application fails to repair airspace enlargement, excessive ROS/RNS, and activation of DAMP signaling, including NF-κB and MAPK signaling; FPS-ZM1 reverses only the infiltration of inflammatory cells and cytokines. Eventually, boosted RAGE expression promotes COPD progression. In this respect, FPS-ZM1 may offer a potential therapeutic application to inhibit inflammation during the development of COPD, where this sustained inflammation is a big hurdle. Taken together, there is a need to verify the affirmative action of FPS-ZM1 against excessive infiltration of inflammatory cells and cytokines in COPD.
Figure Legend Snippet: Proposed mechanism for the effect of FPS-ZM1 in COPD. Exposure to CSE and release of elastase by injured cells during smoking increase ligands, such as S100 calgranulins and HMGB1. The ligands bind to and activate response by RAGE. The engagement of RAGE is linked to the DAMP signaling pathway, notably NF-κB and MAPKs, which overlap considerably with the Nrf2 signaling pathway for RAGE-mediated emphysematous response to stress stimuli in the lung (airspace enlargement, sustained inflammation, excessive ROS/RNS, immune cell infiltration, and epithelial cell death). When mice exposed to PPE or epithelial cells exposed to CSE are treated with FPS-ZM1, RAGE-mediated emphysema and DAMP signaling pathway recover. However, FPS-ZM1 treatment of Nrf2-deficient mice after elastase application fails to repair airspace enlargement, excessive ROS/RNS, and activation of DAMP signaling, including NF-κB and MAPK signaling; FPS-ZM1 reverses only the infiltration of inflammatory cells and cytokines. Eventually, boosted RAGE expression promotes COPD progression. In this respect, FPS-ZM1 may offer a potential therapeutic application to inhibit inflammation during the development of COPD, where this sustained inflammation is a big hurdle. Taken together, there is a need to verify the affirmative action of FPS-ZM1 against excessive infiltration of inflammatory cells and cytokines in COPD.

Techniques Used: Mouse Assay, Activation Assay, Expressing

49) Product Images from "Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells"

Article Title: Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.0277

Macroautophagy mediates PI-induced IκBα degradation (A, B) NCI-H157 cells were transiently transfected with control shRNAs, Lamp2 shRNAs, control siRNAs, or LC3B siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) or MG132 (20 μM) for 8 h. Total cellular extracts were subjected to Western blot analysis for IκBα, Lamp2a, Lamp2, LC3B, and GAPDH. Results are representative of three independent experiments.
Figure Legend Snippet: Macroautophagy mediates PI-induced IκBα degradation (A, B) NCI-H157 cells were transiently transfected with control shRNAs, Lamp2 shRNAs, control siRNAs, or LC3B siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) or MG132 (20 μM) for 8 h. Total cellular extracts were subjected to Western blot analysis for IκBα, Lamp2a, Lamp2, LC3B, and GAPDH. Results are representative of three independent experiments.

Techniques Used: Transfection, Western Blot

50) Product Images from "Decreased DJ-1 Leads to Impaired Nrf2-Regulated Antioxidant Defense and Increased UV-A–Induced Apoptosis in Corneal Endothelial Cells"

Article Title: Decreased DJ-1 Leads to Impaired Nrf2-Regulated Antioxidant Defense and Increased UV-A–Induced Apoptosis in Corneal Endothelial Cells

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.14-14580

The effect of DJ-1 downregulation on Nrf2-associated Cul3 and Keap1 levels. Immunoprecipitation–WB analysis of Cul3/Nrf2 and Keap/Nrf2 levels in non–siRNA-treated, control siRNA-treated, and DJ-1 siRNA-treated HCECi cells. ( A ) Left panel
Figure Legend Snippet: The effect of DJ-1 downregulation on Nrf2-associated Cul3 and Keap1 levels. Immunoprecipitation–WB analysis of Cul3/Nrf2 and Keap/Nrf2 levels in non–siRNA-treated, control siRNA-treated, and DJ-1 siRNA-treated HCECi cells. ( A ) Left panel

Techniques Used: Immunoprecipitation, Western Blot

51) Product Images from "Decreased DJ-1 Leads to Impaired Nrf2-Regulated Antioxidant Defense and Increased UV-A–Induced Apoptosis in Corneal Endothelial Cells"

Article Title: Decreased DJ-1 Leads to Impaired Nrf2-Regulated Antioxidant Defense and Increased UV-A–Induced Apoptosis in Corneal Endothelial Cells

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.14-14580

The effect of DJ-1 downregulation on Nrf2-associated Cul3 and Keap1 levels. Immunoprecipitation–WB analysis of Cul3/Nrf2 and Keap/Nrf2 levels in non–siRNA-treated, control siRNA-treated, and DJ-1 siRNA-treated HCECi cells. ( A ) Left panel
Figure Legend Snippet: The effect of DJ-1 downregulation on Nrf2-associated Cul3 and Keap1 levels. Immunoprecipitation–WB analysis of Cul3/Nrf2 and Keap/Nrf2 levels in non–siRNA-treated, control siRNA-treated, and DJ-1 siRNA-treated HCECi cells. ( A ) Left panel

Techniques Used: Immunoprecipitation, Western Blot

52) Product Images from "KEAP1-NRF2 COMPLEX IN ISCHEMIA-INDUCED HEPATOCELLULAR DAMAGE OF MOUSE LIVER TRANSPLANTS"

Article Title: KEAP1-NRF2 COMPLEX IN ISCHEMIA-INDUCED HEPATOCELLULAR DAMAGE OF MOUSE LIVER TRANSPLANTS

Journal: Journal of hepatology

doi: 10.1016/j.jhep.2013.07.016

Hepatocyte-specific Keap1 deficiency (HKO) promotes anti-apoptotic functions, reduces apoptosis and activates Nrf2-mediated Trx1/Akt/HIF-1 in IR-stressed OLTs. (A) Western analysis of cleaved caspase-3 and Bcl-2/Bcl-xl. Representative of three experiments.
Figure Legend Snippet: Hepatocyte-specific Keap1 deficiency (HKO) promotes anti-apoptotic functions, reduces apoptosis and activates Nrf2-mediated Trx1/Akt/HIF-1 in IR-stressed OLTs. (A) Western analysis of cleaved caspase-3 and Bcl-2/Bcl-xl. Representative of three experiments.

Techniques Used: Western Blot

Inhibition of PI3K disrupts Akt/HIF-1 signaling and recreates liver IRI in Keap1HKO OLTs. Groups of WT or Keap1HKO liver donor mice were pre-treated with Ly294002 or DMSO (-1 h). (A) sALT levels (IU/L): (□) sham; ( ) WT+DMSO; ( ) WT+ Lly294002;
Figure Legend Snippet: Inhibition of PI3K disrupts Akt/HIF-1 signaling and recreates liver IRI in Keap1HKO OLTs. Groups of WT or Keap1HKO liver donor mice were pre-treated with Ly294002 or DMSO (-1 h). (A) sALT levels (IU/L): (□) sham; ( ) WT+DMSO; ( ) WT+ Lly294002;

Techniques Used: Inhibition, Mouse Assay

53) Product Images from "Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells"

Article Title: Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.0277

PI-induced IκBα degradation is associated with macroautophagy (A) NCI-H157 cells were treated with PS-341 (50 nM) or MG132 (20 μM) for the indicated times. Total cellular extracts were subjected to Western blot analysis for Lamp2a, Lamp2, LC3B, and GAPDH. (B) Cells were stimulated with PS-341 (50 nM) for the indicated times. Total RNA was isolated and quantitative real-time PCR for LC3B and GAPDH was performed. Data represent the mean ± SD of triplicate experiments. ** P
Figure Legend Snippet: PI-induced IκBα degradation is associated with macroautophagy (A) NCI-H157 cells were treated with PS-341 (50 nM) or MG132 (20 μM) for the indicated times. Total cellular extracts were subjected to Western blot analysis for Lamp2a, Lamp2, LC3B, and GAPDH. (B) Cells were stimulated with PS-341 (50 nM) for the indicated times. Total RNA was isolated and quantitative real-time PCR for LC3B and GAPDH was performed. Data represent the mean ± SD of triplicate experiments. ** P

Techniques Used: Western Blot, Isolation, Real-time Polymerase Chain Reaction

Macroautophagy mediates PI-induced IκBα degradation (A, B) NCI-H157 cells were transiently transfected with control shRNAs, Lamp2 shRNAs, control siRNAs, or LC3B siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) or MG132 (20 μM) for 8 h. Total cellular extracts were subjected to Western blot analysis for IκBα, Lamp2a, Lamp2, LC3B, and GAPDH. Results are representative of three independent experiments.
Figure Legend Snippet: Macroautophagy mediates PI-induced IκBα degradation (A, B) NCI-H157 cells were transiently transfected with control shRNAs, Lamp2 shRNAs, control siRNAs, or LC3B siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) or MG132 (20 μM) for 8 h. Total cellular extracts were subjected to Western blot analysis for IκBα, Lamp2a, Lamp2, LC3B, and GAPDH. Results are representative of three independent experiments.

Techniques Used: Transfection, Western Blot

54) Product Images from "Attenuation of oxygen fluctuation-induced endoplasmic reticulum stress in human lens epithelial cells"

Article Title: Attenuation of oxygen fluctuation-induced endoplasmic reticulum stress in human lens epithelial cells

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2015.2725

Western blot analysis of the protein levels of HIF)-1α hypoxia-inducible factor in hLECs cultured for 1, 6, 12 and 24 h with 0, 1, 4 and 20% oxygen. HIF, hypoxia-inducible factor; hLECs, human lens epithelial cells.
Figure Legend Snippet: Western blot analysis of the protein levels of HIF)-1α hypoxia-inducible factor in hLECs cultured for 1, 6, 12 and 24 h with 0, 1, 4 and 20% oxygen. HIF, hypoxia-inducible factor; hLECs, human lens epithelial cells.

Techniques Used: Western Blot, Cell Culture

55) Product Images from "Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels"

Article Title: Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels

Journal: Scientific Reports

doi: 10.1038/s41598-018-26049-5

Effect of plasma on chicken sperm mRNA and protein expression. Semen of 60-week-old cocks was exposed to varying plasma potentials for 20 s. Relative mRNA levels of the following genes were measured: ( a ) NOX4 , NRF2 , and KEAP1 ; ( b ) SOD , CAT , and GPx ; ( c ) PRDX1 , PRDX 3, PRDX4 , and PRDX6 ; ( d ) ATP5A1 , ATP5B , ATP5C1 , ATP5F1 , ATP5G1 , ATP5G3 , ATP5H , ATP5I , ATP5J , ATP5J 2, ATP5L , and ATP5S ; and ( e ) AMPKα2 , AMPKβ2 , AMPKγ3 , and mTOR . ( f ) Western blot analysis of protein bands. Uncropped immunoblot scans are presented in Supplementary Figure S1 . The grouping of gels/blots cropped from different gels. All blots were visualized with 5 min exposure time. Relative protein levels of ( g ) NRF2, KEAP1, PRDX4, ( h ) ATP5A, ( i ) p-AMPKα/AMPKα, and ( j ) p-mTOR/mTOR. Values are expressed as the mean ± standard error (n = 3) of three replicates; n represents an individual cock. * p
Figure Legend Snippet: Effect of plasma on chicken sperm mRNA and protein expression. Semen of 60-week-old cocks was exposed to varying plasma potentials for 20 s. Relative mRNA levels of the following genes were measured: ( a ) NOX4 , NRF2 , and KEAP1 ; ( b ) SOD , CAT , and GPx ; ( c ) PRDX1 , PRDX 3, PRDX4 , and PRDX6 ; ( d ) ATP5A1 , ATP5B , ATP5C1 , ATP5F1 , ATP5G1 , ATP5G3 , ATP5H , ATP5I , ATP5J , ATP5J 2, ATP5L , and ATP5S ; and ( e ) AMPKα2 , AMPKβ2 , AMPKγ3 , and mTOR . ( f ) Western blot analysis of protein bands. Uncropped immunoblot scans are presented in Supplementary Figure S1 . The grouping of gels/blots cropped from different gels. All blots were visualized with 5 min exposure time. Relative protein levels of ( g ) NRF2, KEAP1, PRDX4, ( h ) ATP5A, ( i ) p-AMPKα/AMPKα, and ( j ) p-mTOR/mTOR. Values are expressed as the mean ± standard error (n = 3) of three replicates; n represents an individual cock. * p

Techniques Used: Expressing, Western Blot

Cytosine methylation analysis of chicken spermatozoa. Cytosine methylation of ( a ) NRF 2, ( b ) KEAP1 , ( c ) PRDX4 , ( d ) ATP5A1 , ( e ) AMPKα2 , and ( f ) mTOR were analyzed using CyMATE. Lengths of sequenced regions and position of cytosine are shown schematically. The order of the individual sequences of ten clones is listed on the left. 0 represents the control group; 11.7 represents plasma-treated group at 11.7 kV for 20 s; and 27.6 represents plasma-treated group at 27.6 kV for 20 s. The reference sequence is shown in the first line. The sequence is distinguished by circles for CG, squares for CHG, and triangles for CHH. Filled symbols represent methylated cytosine, and open symbols represent unmethylated cytosine. Average methylation levels for CG, CHG, and CHH of ( g ) NRF2 , ( h ) KEAP1 , ( i ) PRDX4 , ( j ) ATP5A1 , ( k ) AMPKα2 , and ( l ) mTOR .
Figure Legend Snippet: Cytosine methylation analysis of chicken spermatozoa. Cytosine methylation of ( a ) NRF 2, ( b ) KEAP1 , ( c ) PRDX4 , ( d ) ATP5A1 , ( e ) AMPKα2 , and ( f ) mTOR were analyzed using CyMATE. Lengths of sequenced regions and position of cytosine are shown schematically. The order of the individual sequences of ten clones is listed on the left. 0 represents the control group; 11.7 represents plasma-treated group at 11.7 kV for 20 s; and 27.6 represents plasma-treated group at 27.6 kV for 20 s. The reference sequence is shown in the first line. The sequence is distinguished by circles for CG, squares for CHG, and triangles for CHH. Filled symbols represent methylated cytosine, and open symbols represent unmethylated cytosine. Average methylation levels for CG, CHG, and CHH of ( g ) NRF2 , ( h ) KEAP1 , ( i ) PRDX4 , ( j ) ATP5A1 , ( k ) AMPKα2 , and ( l ) mTOR .

Techniques Used: Methylation, Clone Assay, Sequencing

56) Product Images from "Expression of UDP-glucuronosyltransferase 1A, nuclear factor erythroid-E2-related factor 2 and Kelch-like ECH-associated protein 1 in colonic mucosa, adenoma and adenocarcinoma tissue"

Article Title: Expression of UDP-glucuronosyltransferase 1A, nuclear factor erythroid-E2-related factor 2 and Kelch-like ECH-associated protein 1 in colonic mucosa, adenoma and adenocarcinoma tissue

Journal: Oncology Letters

doi: 10.3892/ol.2012.850

(A) Nrf2 was expressed in the cytoplasm and nucleus of normal colonic tissues. (B) The adenoma tissues showed strong Nrf2 expression only in the cytoplasm. (C) Strong Nrf2 staining was detected in the adenocarcinoma tissue. x400 magnification. Nrf2, nuclear
Figure Legend Snippet: (A) Nrf2 was expressed in the cytoplasm and nucleus of normal colonic tissues. (B) The adenoma tissues showed strong Nrf2 expression only in the cytoplasm. (C) Strong Nrf2 staining was detected in the adenocarcinoma tissue. x400 magnification. Nrf2, nuclear

Techniques Used: Expressing, Staining

57) Product Images from "Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells"

Article Title: Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.0277

PI-induced IκBα degradation is mediated by Nrf2 up-regulation via both de novo protein synthesis and KEAP1 degradation (A, B) NCI-H157 cells were treated with PS-341 (50 nM) or MG132 (20 μM) for the indicated times. Total cellular extracts were subjected to Western blot analysis for phospho-mTOR (Ser2448) (p-mTOR), sirtuin1, Nrf2, KEAP1, and GAPDH (A). Total RNA was isolated and quantitative real-time PCR for Nrf2 and GAPDH was performed (B). Data represent the mean ± SD of triplicate experiments. ** P
Figure Legend Snippet: PI-induced IκBα degradation is mediated by Nrf2 up-regulation via both de novo protein synthesis and KEAP1 degradation (A, B) NCI-H157 cells were treated with PS-341 (50 nM) or MG132 (20 μM) for the indicated times. Total cellular extracts were subjected to Western blot analysis for phospho-mTOR (Ser2448) (p-mTOR), sirtuin1, Nrf2, KEAP1, and GAPDH (A). Total RNA was isolated and quantitative real-time PCR for Nrf2 and GAPDH was performed (B). Data represent the mean ± SD of triplicate experiments. ** P

Techniques Used: Western Blot, Isolation, Real-time Polymerase Chain Reaction

PI-induced IκBα degradation is associated with macroautophagy (A) NCI-H157 cells were treated with PS-341 (50 nM) or MG132 (20 μM) for the indicated times. Total cellular extracts were subjected to Western blot analysis for Lamp2a, Lamp2, LC3B, and GAPDH. (B) Cells were stimulated with PS-341 (50 nM) for the indicated times. Total RNA was isolated and quantitative real-time PCR for LC3B and GAPDH was performed. Data represent the mean ± SD of triplicate experiments. ** P
Figure Legend Snippet: PI-induced IκBα degradation is associated with macroautophagy (A) NCI-H157 cells were treated with PS-341 (50 nM) or MG132 (20 μM) for the indicated times. Total cellular extracts were subjected to Western blot analysis for Lamp2a, Lamp2, LC3B, and GAPDH. (B) Cells were stimulated with PS-341 (50 nM) for the indicated times. Total RNA was isolated and quantitative real-time PCR for LC3B and GAPDH was performed. Data represent the mean ± SD of triplicate experiments. ** P

Techniques Used: Western Blot, Isolation, Real-time Polymerase Chain Reaction

Macroautophagy mediates PI-induced IκBα degradation (A, B) NCI-H157 cells were transiently transfected with control shRNAs, Lamp2 shRNAs, control siRNAs, or LC3B siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) or MG132 (20 μM) for 8 h. Total cellular extracts were subjected to Western blot analysis for IκBα, Lamp2a, Lamp2, LC3B, and GAPDH. Results are representative of three independent experiments.
Figure Legend Snippet: Macroautophagy mediates PI-induced IκBα degradation (A, B) NCI-H157 cells were transiently transfected with control shRNAs, Lamp2 shRNAs, control siRNAs, or LC3B siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) or MG132 (20 μM) for 8 h. Total cellular extracts were subjected to Western blot analysis for IκBα, Lamp2a, Lamp2, LC3B, and GAPDH. Results are representative of three independent experiments.

Techniques Used: Transfection, Western Blot

PI activates IκB/NF-κB pathway (A) NCI-H157 and A549 cells were treated with PS-341 (50 nM) or MG132 (20 μM) for the indicated times. Total cellular extracts were subjected to Western blot analysis for IκBα and GAPDH. (B) NCI-H157 cells were treated with PS-341 or MG132 for the indicated times. Nuclear proteins were extracted and subjected to Western blot analysis for p65 and Lamin A/C. TNF-α (10 ng/mL, 60 min) was used as a positive control. (C) Both cell lines were stimulated with PS-341 or MG132 for 0, 6, 8, 12, or 24 h. Total RNA was isolated and quantitative real-time PCR for COX-2 and GAPDH was performed. Data represent the mean ± SD of triplicate experiments. ** P
Figure Legend Snippet: PI activates IκB/NF-κB pathway (A) NCI-H157 and A549 cells were treated with PS-341 (50 nM) or MG132 (20 μM) for the indicated times. Total cellular extracts were subjected to Western blot analysis for IκBα and GAPDH. (B) NCI-H157 cells were treated with PS-341 or MG132 for the indicated times. Nuclear proteins were extracted and subjected to Western blot analysis for p65 and Lamin A/C. TNF-α (10 ng/mL, 60 min) was used as a positive control. (C) Both cell lines were stimulated with PS-341 or MG132 for 0, 6, 8, 12, or 24 h. Total RNA was isolated and quantitative real-time PCR for COX-2 and GAPDH was performed. Data represent the mean ± SD of triplicate experiments. ** P

Techniques Used: Western Blot, Positive Control, Isolation, Real-time Polymerase Chain Reaction

PI-induced Nrf2 activation suppresses PI-induced cell death (A) NCI-H157 cells were transiently transfected with control siRNAs or Nrf2 siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) for 24 h. Total cellular extracts were subjected to Western blot analysis for PARP, Nrf2, and GAPDH. (B) Cells were infected with adenovirus vectors expressing WT-Nrf2 or DN-Nrf2. After 48 h, the cells were stimulated with PS-341 (50 nM) for 24 h. Cell viability was determined by MTT assay. Data represent the mean ± SD of triplicate experiments. ** P
Figure Legend Snippet: PI-induced Nrf2 activation suppresses PI-induced cell death (A) NCI-H157 cells were transiently transfected with control siRNAs or Nrf2 siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) for 24 h. Total cellular extracts were subjected to Western blot analysis for PARP, Nrf2, and GAPDH. (B) Cells were infected with adenovirus vectors expressing WT-Nrf2 or DN-Nrf2. After 48 h, the cells were stimulated with PS-341 (50 nM) for 24 h. Cell viability was determined by MTT assay. Data represent the mean ± SD of triplicate experiments. ** P

Techniques Used: Activation Assay, Transfection, Western Blot, Infection, Expressing, MTT Assay

58) Product Images from "High glucose induces renal tubular epithelial injury via Sirt1/NF-kappaB/microR-29/Keap1 signal pathway"

Article Title: High glucose induces renal tubular epithelial injury via Sirt1/NF-kappaB/microR-29/Keap1 signal pathway

Journal: Journal of Translational Medicine

doi: 10.1186/s12967-015-0710-y

Effect of high glucose on renal tubule epithelia cell of HK-2 in vitro. Cells were triggered with doses of glucose (5.5, 15, 30 and 45) for 48 h. a Sirt1 activity was assessed. b NF-κB transcription activity was evaluated using luciferase reporter gene assay. c miR-29 expression was determined. d Western blot was performed to assess Keap1, GST, NQO1 and nuclear Nrf-2 expression. e Cell viability was evaluated using MTT assay. Data were presented as mean ± S.D. **P
Figure Legend Snippet: Effect of high glucose on renal tubule epithelia cell of HK-2 in vitro. Cells were triggered with doses of glucose (5.5, 15, 30 and 45) for 48 h. a Sirt1 activity was assessed. b NF-κB transcription activity was evaluated using luciferase reporter gene assay. c miR-29 expression was determined. d Western blot was performed to assess Keap1, GST, NQO1 and nuclear Nrf-2 expression. e Cell viability was evaluated using MTT assay. Data were presented as mean ± S.D. **P

Techniques Used: In Vitro, Activity Assay, Luciferase, Reporter Gene Assay, Expressing, Western Blot, MTT Assay

59) Product Images from "Bovine Herpesvirus 1 Productive Infection Led to Inactivation of Nrf2 Signaling through Diverse Approaches"

Article Title: Bovine Herpesvirus 1 Productive Infection Led to Inactivation of Nrf2 Signaling through Diverse Approaches

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2019/4957878

BoHV-1 infection altered the localization of nuclear Nrf2 protein. MDBK cells in 2-well chamber slides were mock infected (a) or infected with BoHV-1 (MOI = 0.1) (b) for 24 hours. After three washings with PBS, cells were fixed with 4% formaldehyde, and Nrf2 was detected by IFA using the Nrf2 antibody (1 : 500) and LaminA/C antibody (1 : 500). DAPI staining was used to stain nuclear DNA. Images were obtained by performing confocal microscopy (Leica). These images are representative of three independent experiments. (c) Zoom-in cells showing typical dot-like staining. (d) The percentage of dot-like staining-positive cells among ~400 cells was estimated from photos derived from three independent experiments. Scale bar: 200 μ M.
Figure Legend Snippet: BoHV-1 infection altered the localization of nuclear Nrf2 protein. MDBK cells in 2-well chamber slides were mock infected (a) or infected with BoHV-1 (MOI = 0.1) (b) for 24 hours. After three washings with PBS, cells were fixed with 4% formaldehyde, and Nrf2 was detected by IFA using the Nrf2 antibody (1 : 500) and LaminA/C antibody (1 : 500). DAPI staining was used to stain nuclear DNA. Images were obtained by performing confocal microscopy (Leica). These images are representative of three independent experiments. (c) Zoom-in cells showing typical dot-like staining. (d) The percentage of dot-like staining-positive cells among ~400 cells was estimated from photos derived from three independent experiments. Scale bar: 200 μ M.

Techniques Used: Infection, Immunofluorescence, Staining, Confocal Microscopy, Derivative Assay

BoHV-1 infection altered the accumulation of Nrf2 in the nucleus. (a) MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1 (MOI = 0.1) for 24 h, the cell cultures were collected for extracting nuclear proteins using commercial nuclear protein purification kit (Beyotime Biotechnology, cat# P0027). Nrf2 was detected by Western blot using the antibody against Nrf2 (Abcam, cat# ab137550). LaminA/C was detected and used as protein loading control. (b) Protein fractions of both the cytosol and nucleus were subjected to Western blots using the antibody against β -tubulin. (c) MDBK cells in 60 mm dishes were mock treated or treated with Trolox (1 mM) for 2 h; the cell lysates were collected for the purification of nuclear proteins and cytosol proteins using commercial nuclear protein purification kit (Beyotime Biotechnology, cat# P0027). Nrf2 was detected by Western blot using the antibody against Nrf2 (Abcam, cat# ab137550). LaminA/C and β -tubulin were detected to characterize whether each fraction was contaminated. These images are representative of three independent experiments.
Figure Legend Snippet: BoHV-1 infection altered the accumulation of Nrf2 in the nucleus. (a) MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1 (MOI = 0.1) for 24 h, the cell cultures were collected for extracting nuclear proteins using commercial nuclear protein purification kit (Beyotime Biotechnology, cat# P0027). Nrf2 was detected by Western blot using the antibody against Nrf2 (Abcam, cat# ab137550). LaminA/C was detected and used as protein loading control. (b) Protein fractions of both the cytosol and nucleus were subjected to Western blots using the antibody against β -tubulin. (c) MDBK cells in 60 mm dishes were mock treated or treated with Trolox (1 mM) for 2 h; the cell lysates were collected for the purification of nuclear proteins and cytosol proteins using commercial nuclear protein purification kit (Beyotime Biotechnology, cat# P0027). Nrf2 was detected by Western blot using the antibody against Nrf2 (Abcam, cat# ab137550). LaminA/C and β -tubulin were detected to characterize whether each fraction was contaminated. These images are representative of three independent experiments.

Techniques Used: Infection, Protein Purification, Western Blot, Purification

BoHV-1 affected the association between Nrf2 and LaminA/C. MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1 for 24 h. Nucleus proteins were purified using commercial nuclear protein purification kit (Beyotime Biotechnology, cat# P0027). IP was performed using antibodies against Nrf2 (a), LaminA/C (b), Nrf2 and unrelated IgG (c), and LaminA/C and IgG (d) (R: rabbit; M: mouse). Then, Western blots were performed using corresponding antibodies. (e) MDBK cells in 60 mm dishes were mock treated or treated with Trolox (1 mM) for 2 h. Nucleus proteins were purified using commercial nuclear protein purification kit (Beyotime Biotechnology, cat# P0027), and LaminA/C was detected by Western blots.
Figure Legend Snippet: BoHV-1 affected the association between Nrf2 and LaminA/C. MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1 for 24 h. Nucleus proteins were purified using commercial nuclear protein purification kit (Beyotime Biotechnology, cat# P0027). IP was performed using antibodies against Nrf2 (a), LaminA/C (b), Nrf2 and unrelated IgG (c), and LaminA/C and IgG (d) (R: rabbit; M: mouse). Then, Western blots were performed using corresponding antibodies. (e) MDBK cells in 60 mm dishes were mock treated or treated with Trolox (1 mM) for 2 h. Nucleus proteins were purified using commercial nuclear protein purification kit (Beyotime Biotechnology, cat# P0027), and LaminA/C was detected by Western blots.

Techniques Used: Infection, Purification, Protein Purification, Western Blot

60) Product Images from "NADPH oxidase 4 deficiency increases tubular cell death during acute ischemic reperfusion injury"

Article Title: NADPH oxidase 4 deficiency increases tubular cell death during acute ischemic reperfusion injury

Journal: Scientific Reports

doi: 10.1038/srep38598

Overexpression of active NRF2 partially prevents NOX4 deletion-associated pro-apoptotic phenotype. ( A ) Real time PCR analysis of NRF2 mRNA and ( B ) Western blot analysis of NRF2 protein and β-actin performed on WT MEF cells transfected or not with active NRF2. Representative images are shown. Results are expressed as ratio of relative quantity over the mean value obtained in WT control cells ± SEM, (n = 6), ns p > 0.05, * or # p
Figure Legend Snippet: Overexpression of active NRF2 partially prevents NOX4 deletion-associated pro-apoptotic phenotype. ( A ) Real time PCR analysis of NRF2 mRNA and ( B ) Western blot analysis of NRF2 protein and β-actin performed on WT MEF cells transfected or not with active NRF2. Representative images are shown. Results are expressed as ratio of relative quantity over the mean value obtained in WT control cells ± SEM, (n = 6), ns p > 0.05, * or # p

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Transfection

NOX4 regulates NRF2 by modifying KEAP1 oxidation. ( A–D ) Western blot analysis of KEAP1 oxidation performed on kidney cortex from WT and NOX4 KO sham mice, in non-reducing and reduced conditions. Representative images ( A,C ) and high molecular weight oxidized KEAP1 densitometric quantifications (WT = 6 and NOX4KO = 6) ( B,D ) are shown respectively in non-reduced ( A,B ) and reduced conditions ( C,D ). Results are expressed as the mean of individual densitometric values over the mean of densitometric value obtained in WT animals ± SEM, ns p > 0.05, *p
Figure Legend Snippet: NOX4 regulates NRF2 by modifying KEAP1 oxidation. ( A–D ) Western blot analysis of KEAP1 oxidation performed on kidney cortex from WT and NOX4 KO sham mice, in non-reducing and reduced conditions. Representative images ( A,C ) and high molecular weight oxidized KEAP1 densitometric quantifications (WT = 6 and NOX4KO = 6) ( B,D ) are shown respectively in non-reduced ( A,B ) and reduced conditions ( C,D ). Results are expressed as the mean of individual densitometric values over the mean of densitometric value obtained in WT animals ± SEM, ns p > 0.05, *p

Techniques Used: Western Blot, Mouse Assay, Molecular Weight

NRF2 and NRF2 target genes expressions are downregulated in NOX4 KO mice under IRI. ( A,B ) Western blot analysis of NRF2 performed on kidney cortex from NOX4 and WT mice after IR. Representative picture and densitometric quantification (WT, n = 3; NOX4 KO, n = 5) of Western blots are shown. Results are expressed as the mean of individual densitometric values of the mean of densitometric value obtained in WT animals ± SEM, ns p > 0.05, *p
Figure Legend Snippet: NRF2 and NRF2 target genes expressions are downregulated in NOX4 KO mice under IRI. ( A,B ) Western blot analysis of NRF2 performed on kidney cortex from NOX4 and WT mice after IR. Representative picture and densitometric quantification (WT, n = 3; NOX4 KO, n = 5) of Western blots are shown. Results are expressed as the mean of individual densitometric values of the mean of densitometric value obtained in WT animals ± SEM, ns p > 0.05, *p

Techniques Used: Mouse Assay, Western Blot

61) Product Images from "The Cinnamon-derived Dietary Factor Cinnamic Aldehyde Activates the Nrf2-dependent Antioxidant Response in Human Epithelial Colon Cells"

Article Title: The Cinnamon-derived Dietary Factor Cinnamic Aldehyde Activates the Nrf2-dependent Antioxidant Response in Human Epithelial Colon Cells

Journal: Molecules (Basel, Switzerland)

doi: 10.3390/molecules15053338

Dose response of Nrf2 transcriptional activation by cinnamic aldehyde and an ethanolic cinnamon extract in MDA-MB231 breast carcinoma and HCT116 colon carcinoma cells. (a–b) MDA-MB-231 cells (panel a) or HCT116 (panel b) were cotransfected with plasmids containing a GST-ARE-firefly luciferase reporter gene and expression plasmids for the Nrf2 and Keap1 proteins. A plasmid encoding renilla luciferase, driven by the herpes simplex virus thymidine kinase promoter, was included in all transfections to normalize transfection efficiency. Twenty-four hours post-transfection, cells were dosed with the indicated concentrations of each compound (tBHQ: 50μM) for 16h. Firefly and renilla luciferase activity was measured and is expressed as relative activity (F/R) compared to untreated control ( p
Figure Legend Snippet: Dose response of Nrf2 transcriptional activation by cinnamic aldehyde and an ethanolic cinnamon extract in MDA-MB231 breast carcinoma and HCT116 colon carcinoma cells. (a–b) MDA-MB-231 cells (panel a) or HCT116 (panel b) were cotransfected with plasmids containing a GST-ARE-firefly luciferase reporter gene and expression plasmids for the Nrf2 and Keap1 proteins. A plasmid encoding renilla luciferase, driven by the herpes simplex virus thymidine kinase promoter, was included in all transfections to normalize transfection efficiency. Twenty-four hours post-transfection, cells were dosed with the indicated concentrations of each compound (tBHQ: 50μM) for 16h. Firefly and renilla luciferase activity was measured and is expressed as relative activity (F/R) compared to untreated control ( p

Techniques Used: Activation Assay, Multiple Displacement Amplification, Luciferase, Expressing, Plasmid Preparation, Transfection, Activity Assay

62) Product Images from "Oxidative stress and cell damage in a model of precancerous lesions and advanced hepatocellular carcinoma in rats"

Article Title: Oxidative stress and cell damage in a model of precancerous lesions and advanced hepatocellular carcinoma in rats

Journal: Toxicology Reports

doi: 10.1016/j.toxrep.2014.11.015

Western blot analysis of NQO1, Keap1, Nrf2, HP73 and HP72. Protein from liver extracts was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. (A) Representative images. (B) Densitometric quantification. Values are expressed as means ± SD ( n = 5). a p
Figure Legend Snippet: Western blot analysis of NQO1, Keap1, Nrf2, HP73 and HP72. Protein from liver extracts was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. (A) Representative images. (B) Densitometric quantification. Values are expressed as means ± SD ( n = 5). a p

Techniques Used: Western Blot, Polyacrylamide Gel Electrophoresis

63) Product Images from "miR-200a Regulates Nrf2 Activation by Targeting Keap1 mRNA in Breast Cancer Cells *"

Article Title: miR-200a Regulates Nrf2 Activation by Targeting Keap1 mRNA in Breast Cancer Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.275495

miR-200a promotes Nrf2 activation by targeting Keap1 mRNA. A , miR-200a targets Keap1 mRNA, resulting in Keap1 mRNA destabilization. The Keap1 mRNA stability assay was performed in MDA-MB-231 and Hs578T cell lines transfected with miR-200a expression vector
Figure Legend Snippet: miR-200a promotes Nrf2 activation by targeting Keap1 mRNA. A , miR-200a targets Keap1 mRNA, resulting in Keap1 mRNA destabilization. The Keap1 mRNA stability assay was performed in MDA-MB-231 and Hs578T cell lines transfected with miR-200a expression vector

Techniques Used: Activation Assay, Stability Assay, Multiple Displacement Amplification, Transfection, Expressing, Plasmid Preparation

miR-200a targets Keap1 in breast cancer cell lines. A , array profiling of 88 miRs comparing expression in MCF-10A and MDA-MB-231 cell lines. The scatter plot shows -fold change in miR expression between cell lines (log 10 (2 Δ Ct )). The middle line
Figure Legend Snippet: miR-200a targets Keap1 in breast cancer cell lines. A , array profiling of 88 miRs comparing expression in MCF-10A and MDA-MB-231 cell lines. The scatter plot shows -fold change in miR expression between cell lines (log 10 (2 Δ Ct )). The middle line

Techniques Used: Expressing, Multiple Displacement Amplification

Treatment with SAHA reduces anchorage-independent cell growth in breast cancer cell lines and impacts miR-200a/Keap1/Nrf2 pathway in vivo . A , Western blotting showing Nrf2 protein expression in Nrf2-myc vector- or control vector-transfected MDA-MB-231
Figure Legend Snippet: Treatment with SAHA reduces anchorage-independent cell growth in breast cancer cell lines and impacts miR-200a/Keap1/Nrf2 pathway in vivo . A , Western blotting showing Nrf2 protein expression in Nrf2-myc vector- or control vector-transfected MDA-MB-231

Techniques Used: In Vivo, Western Blot, Expressing, Plasmid Preparation, Transfection, Multiple Displacement Amplification

Epigenetic therapy restores Nrf2 activity in breast cancer cell lines through miR-200a re-expression and Keap1 down-regulation. A , SAHA treatment of breast cancer cell lines results in miR-200a re-expression. miR-200a levels in SAHA-treated Hs578T cells
Figure Legend Snippet: Epigenetic therapy restores Nrf2 activity in breast cancer cell lines through miR-200a re-expression and Keap1 down-regulation. A , SAHA treatment of breast cancer cell lines results in miR-200a re-expression. miR-200a levels in SAHA-treated Hs578T cells

Techniques Used: Activity Assay, Expressing

64) Product Images from "Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling"

Article Title: Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling

Journal: The FASEB Journal

doi: 10.1096/fj.201601155R

Proposed mechanism for the effect of FPS-ZM1 in COPD. Exposure to CSE and release of elastase by injured cells during smoking increase ligands, such as S100 calgranulins and HMGB1. The ligands bind to and activate response by RAGE. The engagement of RAGE is linked to the DAMP signaling pathway, notably NF-κB and MAPKs, which overlap considerably with the Nrf2 signaling pathway for RAGE-mediated emphysematous response to stress stimuli in the lung (airspace enlargement, sustained inflammation, excessive ROS/RNS, immune cell infiltration, and epithelial cell death). When mice exposed to PPE or epithelial cells exposed to CSE are treated with FPS-ZM1, RAGE-mediated emphysema and DAMP signaling pathway recover. However, FPS-ZM1 treatment of Nrf2-deficient mice after elastase application fails to repair airspace enlargement, excessive ROS/RNS, and activation of DAMP signaling, including NF-κB and MAPK signaling; FPS-ZM1 reverses only the infiltration of inflammatory cells and cytokines. Eventually, boosted RAGE expression promotes COPD progression. In this respect, FPS-ZM1 may offer a potential therapeutic application to inhibit inflammation during the development of COPD, where this sustained inflammation is a big hurdle. Taken together, there is a need to verify the affirmative action of FPS-ZM1 against excessive infiltration of inflammatory cells and cytokines in COPD.
Figure Legend Snippet: Proposed mechanism for the effect of FPS-ZM1 in COPD. Exposure to CSE and release of elastase by injured cells during smoking increase ligands, such as S100 calgranulins and HMGB1. The ligands bind to and activate response by RAGE. The engagement of RAGE is linked to the DAMP signaling pathway, notably NF-κB and MAPKs, which overlap considerably with the Nrf2 signaling pathway for RAGE-mediated emphysematous response to stress stimuli in the lung (airspace enlargement, sustained inflammation, excessive ROS/RNS, immune cell infiltration, and epithelial cell death). When mice exposed to PPE or epithelial cells exposed to CSE are treated with FPS-ZM1, RAGE-mediated emphysema and DAMP signaling pathway recover. However, FPS-ZM1 treatment of Nrf2-deficient mice after elastase application fails to repair airspace enlargement, excessive ROS/RNS, and activation of DAMP signaling, including NF-κB and MAPK signaling; FPS-ZM1 reverses only the infiltration of inflammatory cells and cytokines. Eventually, boosted RAGE expression promotes COPD progression. In this respect, FPS-ZM1 may offer a potential therapeutic application to inhibit inflammation during the development of COPD, where this sustained inflammation is a big hurdle. Taken together, there is a need to verify the affirmative action of FPS-ZM1 against excessive infiltration of inflammatory cells and cytokines in COPD.

Techniques Used: Mouse Assay, Activation Assay, Expressing

Effect of FPS-ZM1 on activation of DAMP signaling in PPE-induced experimental COPD. A ) IKKα, IKKβ, and phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) were determined by Western blot analysis. B ) JNK/ERK/p38 MAPK activation (phosphorylation) was determined by Western blot with the appropriate antibodies. The target protein bands were subjected to densitometric analysis normalized with β-actin. C ) The canonical–noncanonical Rel/p65 NF-κB activation and MAPK activation and its correlation to Nrf2 translocation from the cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for the nuclear protein loading control, and β-actin was used for the cytoplasmic protein loading control. The corresponding density ratio was determined by the average intensity of the bands. Ctl, control group; ns, not significant. * P
Figure Legend Snippet: Effect of FPS-ZM1 on activation of DAMP signaling in PPE-induced experimental COPD. A ) IKKα, IKKβ, and phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) were determined by Western blot analysis. B ) JNK/ERK/p38 MAPK activation (phosphorylation) was determined by Western blot with the appropriate antibodies. The target protein bands were subjected to densitometric analysis normalized with β-actin. C ) The canonical–noncanonical Rel/p65 NF-κB activation and MAPK activation and its correlation to Nrf2 translocation from the cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for the nuclear protein loading control, and β-actin was used for the cytoplasmic protein loading control. The corresponding density ratio was determined by the average intensity of the bands. Ctl, control group; ns, not significant. * P

Techniques Used: Activation Assay, Western Blot, Translocation Assay, CTL Assay

Effect of FPS-ZM1 on CSE-induced activation of DAMP signaling in MLE-12 cells. A – C ) After exposure to CSE in the presence or absence of FPS-ZM1 for 24 h, Western blot analysis was perfomed ( A ), and the expression levels of mRAGE in lung homogenates ( B ) and of circulating sRAGE ( C ) were determined by ELISA. D , E ) IKKα, IKKβ, and the phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) ( D ) and the phosphorylation of JNK/ERK/p38 MAPK activation ( E ) were detected with the appropriate antibodies. β-Actin, total RelA/p65, total JNK1/2, total ERK1/2, and total p38 were used for loading controls. F ) Correlation to Nrf2 translocation from cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for nuclear protein loading control, and β-actin was used for cytoplasmic protein loading control. The activation of DAMP signaling was inhibited by FPS-ZM1 treatment. Results are combined data from 3 independent experiments. Ctl, control group; ns, not significant. * P
Figure Legend Snippet: Effect of FPS-ZM1 on CSE-induced activation of DAMP signaling in MLE-12 cells. A – C ) After exposure to CSE in the presence or absence of FPS-ZM1 for 24 h, Western blot analysis was perfomed ( A ), and the expression levels of mRAGE in lung homogenates ( B ) and of circulating sRAGE ( C ) were determined by ELISA. D , E ) IKKα, IKKβ, and the phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) ( D ) and the phosphorylation of JNK/ERK/p38 MAPK activation ( E ) were detected with the appropriate antibodies. β-Actin, total RelA/p65, total JNK1/2, total ERK1/2, and total p38 were used for loading controls. F ) Correlation to Nrf2 translocation from cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for nuclear protein loading control, and β-actin was used for cytoplasmic protein loading control. The activation of DAMP signaling was inhibited by FPS-ZM1 treatment. Results are combined data from 3 independent experiments. Ctl, control group; ns, not significant. * P

Techniques Used: Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Translocation Assay, CTL Assay

65) Product Images from "MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation"

Article Title: MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation

Journal: Scientific Reports

doi: 10.1038/s41598-018-27123-8

ROS production and antioxidant activity of plasma-treated SCs. Chicken SCs were exposed to 22.0 kV of non-thermal plasma for 120 s. ( A ) Imaging of SCs stained with DCFDA/MitoSOX Red/DAPI. Intracellular ROS production was detected by DCFDA staining and mitochondrial superoxide was detected by MitoSOX Red staining. DCFDA: green fluorescence; MitoSOX Red: red fluorescence; DAPI: blue fluorescence. Scale bar: 50 μm. ( B ) Relative fluorescence intensity for DCFDA staining. ( C ) Relative fluorescence intensity for MitoSOX Red staining. ( D ) Total ROS levels in SCs. ( E ) MDA level in SCs. Activities of ( F ) SOD, ( G ) CAT, and ( H ) GPx in SCs. Relative mRNA levels of ( I ) NOX4 , NRF2 , and KEAP1 , and ( J ) SOD , CAT , GPx , and PRDX4 . Data are represented as the mean ± SD (n = 3 per group). * p
Figure Legend Snippet: ROS production and antioxidant activity of plasma-treated SCs. Chicken SCs were exposed to 22.0 kV of non-thermal plasma for 120 s. ( A ) Imaging of SCs stained with DCFDA/MitoSOX Red/DAPI. Intracellular ROS production was detected by DCFDA staining and mitochondrial superoxide was detected by MitoSOX Red staining. DCFDA: green fluorescence; MitoSOX Red: red fluorescence; DAPI: blue fluorescence. Scale bar: 50 μm. ( B ) Relative fluorescence intensity for DCFDA staining. ( C ) Relative fluorescence intensity for MitoSOX Red staining. ( D ) Total ROS levels in SCs. ( E ) MDA level in SCs. Activities of ( F ) SOD, ( G ) CAT, and ( H ) GPx in SCs. Relative mRNA levels of ( I ) NOX4 , NRF2 , and KEAP1 , and ( J ) SOD , CAT , GPx , and PRDX4 . Data are represented as the mean ± SD (n = 3 per group). * p

Techniques Used: Antioxidant Activity Assay, Imaging, Staining, Fluorescence, Multiple Displacement Amplification

Chicken SC protein expression. ( A . Relative protein levels of ( B ) NRF2, KEAP1, PRDX4, ( C ) ATP5A, ( D ) p-AMPKα/AMPKα, and ( E ) p-mTOR/mTOR in SCs exposed to plasma. ( F . Relative protein levels of ( G ) ATP5A, ( H ) p-AMPKα/AMPKα, and ( I . Data are represented as the mean ± SD (n = 3 per group). * p
Figure Legend Snippet: Chicken SC protein expression. ( A . Relative protein levels of ( B ) NRF2, KEAP1, PRDX4, ( C ) ATP5A, ( D ) p-AMPKα/AMPKα, and ( E ) p-mTOR/mTOR in SCs exposed to plasma. ( F . Relative protein levels of ( G ) ATP5A, ( H ) p-AMPKα/AMPKα, and ( I . Data are represented as the mean ± SD (n = 3 per group). * p

Techniques Used: Expressing

66) Product Images from "EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation"

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

Journal: Autophagy

doi: 10.1080/15548627.2018.1536530

EBV reduces mitochondrial biogenesis in differentiating monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) CYCS and (b) for NRF1 and TFAM expression by western blot. ACTB was used as a loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of CYCS:ACTB, NRF1:ACTB and TFAM:ACTB of 3 different experiments. * P value
Figure Legend Snippet: EBV reduces mitochondrial biogenesis in differentiating monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) CYCS and (b) for NRF1 and TFAM expression by western blot. ACTB was used as a loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of CYCS:ACTB, NRF1:ACTB and TFAM:ACTB of 3 different experiments. * P value

Techniques Used: Cell Culture, Expressing, Western Blot

67) Product Images from "Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells"

Article Title: Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.0277

PI-induced Nrf2 activation suppresses PI-induced cell death (A) NCI-H157 cells were transiently transfected with control siRNAs or Nrf2 siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) for 24 h. Total cellular extracts were subjected to Western blot analysis for PARP, Nrf2, and GAPDH. (B) Cells were infected with adenovirus vectors expressing WT-Nrf2 or DN-Nrf2. After 48 h, the cells were stimulated with PS-341 (50 nM) for 24 h. Cell viability was determined by MTT assay. Data represent the mean ± SD of triplicate experiments. ** P
Figure Legend Snippet: PI-induced Nrf2 activation suppresses PI-induced cell death (A) NCI-H157 cells were transiently transfected with control siRNAs or Nrf2 siRNAs. Forty-eight hours after transfection, the cells were treated with PS-341 (50 nM) for 24 h. Total cellular extracts were subjected to Western blot analysis for PARP, Nrf2, and GAPDH. (B) Cells were infected with adenovirus vectors expressing WT-Nrf2 or DN-Nrf2. After 48 h, the cells were stimulated with PS-341 (50 nM) for 24 h. Cell viability was determined by MTT assay. Data represent the mean ± SD of triplicate experiments. ** P

Techniques Used: Activation Assay, Transfection, Western Blot, Infection, Expressing, MTT Assay

68) Product Images from "Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling"

Article Title: Blockade of RAGE ameliorates elastase-induced emphysema development and progression via RAGE-DAMP signaling

Journal: The FASEB Journal

doi: 10.1096/fj.201601155R

Effect of FPS-ZM1 on activation of DAMP signaling in PPE-induced experimental COPD. A ) IKKα, IKKβ, and phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) were determined by Western blot analysis. B ) JNK/ERK/p38 MAPK activation (phosphorylation) was determined by Western blot with the appropriate antibodies. The target protein bands were subjected to densitometric analysis normalized with β-actin. C ) The canonical–noncanonical Rel/p65 NF-κB activation and MAPK activation and its correlation to Nrf2 translocation from the cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for the nuclear protein loading control, and β-actin was used for the cytoplasmic protein loading control. The corresponding density ratio was determined by the average intensity of the bands. Ctl, control group; ns, not significant. * P
Figure Legend Snippet: Effect of FPS-ZM1 on activation of DAMP signaling in PPE-induced experimental COPD. A ) IKKα, IKKβ, and phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) were determined by Western blot analysis. B ) JNK/ERK/p38 MAPK activation (phosphorylation) was determined by Western blot with the appropriate antibodies. The target protein bands were subjected to densitometric analysis normalized with β-actin. C ) The canonical–noncanonical Rel/p65 NF-κB activation and MAPK activation and its correlation to Nrf2 translocation from the cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for the nuclear protein loading control, and β-actin was used for the cytoplasmic protein loading control. The corresponding density ratio was determined by the average intensity of the bands. Ctl, control group; ns, not significant. * P

Techniques Used: Activation Assay, Western Blot, Translocation Assay, CTL Assay

Effect of FPS-ZM1 on CSE-induced activation of DAMP signaling in MLE-12 cells. A – C ) After exposure to CSE in the presence or absence of FPS-ZM1 for 24 h, Western blot analysis was perfomed ( A ), and the expression levels of mRAGE in lung homogenates ( B ) and of circulating sRAGE ( C ) were determined by ELISA. D , E ) IKKα, IKKβ, and the phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) ( D ) and the phosphorylation of JNK/ERK/p38 MAPK activation ( E ) were detected with the appropriate antibodies. β-Actin, total RelA/p65, total JNK1/2, total ERK1/2, and total p38 were used for loading controls. F ) Correlation to Nrf2 translocation from cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for nuclear protein loading control, and β-actin was used for cytoplasmic protein loading control. The activation of DAMP signaling was inhibited by FPS-ZM1 treatment. Results are combined data from 3 independent experiments. Ctl, control group; ns, not significant. * P
Figure Legend Snippet: Effect of FPS-ZM1 on CSE-induced activation of DAMP signaling in MLE-12 cells. A – C ) After exposure to CSE in the presence or absence of FPS-ZM1 for 24 h, Western blot analysis was perfomed ( A ), and the expression levels of mRAGE in lung homogenates ( B ) and of circulating sRAGE ( C ) were determined by ELISA. D , E ) IKKα, IKKβ, and the phosphorylation of Rel/p65 at 2 sites (Ser276 and Ser536) ( D ) and the phosphorylation of JNK/ERK/p38 MAPK activation ( E ) were detected with the appropriate antibodies. β-Actin, total RelA/p65, total JNK1/2, total ERK1/2, and total p38 were used for loading controls. F ) Correlation to Nrf2 translocation from cytoplasm into the nucleus was measured by Western blot analysis. Lamin A/C was used for nuclear protein loading control, and β-actin was used for cytoplasmic protein loading control. The activation of DAMP signaling was inhibited by FPS-ZM1 treatment. Results are combined data from 3 independent experiments. Ctl, control group; ns, not significant. * P

Techniques Used: Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Translocation Assay, CTL Assay

69) Product Images from "Schisandrol B protects against acetaminophen-induced acute hepatotoxicity in mice via activation of the NRF2/ARE signaling pathway"

Article Title: Schisandrol B protects against acetaminophen-induced acute hepatotoxicity in mice via activation of the NRF2/ARE signaling pathway

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2015.120

The effects of SolB on activation and expression of NRF2 in vivo . (A) qRT-PCR analysis was performed to measure the gene expression of Nrf2, and the data are expressed as the mean±SEM ( n =6). (B–C) Western blotting was used to measure nuclear
Figure Legend Snippet: The effects of SolB on activation and expression of NRF2 in vivo . (A) qRT-PCR analysis was performed to measure the gene expression of Nrf2, and the data are expressed as the mean±SEM ( n =6). (B–C) Western blotting was used to measure nuclear

Techniques Used: Activation Assay, Expressing, In Vivo, Quantitative RT-PCR, Western Blot

Role of NRF2 signaling and its downstream proteins in SolB-mediated protection against APAP-induced liver toxicity. (A) Western blotting was used to measure the protein expression of GSR, GSS, GCLC, GCLM, GST-α, GST-μ, GST-π, NQO1,
Figure Legend Snippet: Role of NRF2 signaling and its downstream proteins in SolB-mediated protection against APAP-induced liver toxicity. (A) Western blotting was used to measure the protein expression of GSR, GSS, GCLC, GCLM, GST-α, GST-μ, GST-π, NQO1,

Techniques Used: Western Blot, Expressing

Effects of SolB on cell viability and NRF2 activation. (A) An MTT assay was used to measure HepG2 cell viability after treatment with SolB at 2.5 to 160 μmol/L. (B) A luciferase reporter assay was used to measure the effect of SolB on NRF2 activation.
Figure Legend Snippet: Effects of SolB on cell viability and NRF2 activation. (A) An MTT assay was used to measure HepG2 cell viability after treatment with SolB at 2.5 to 160 μmol/L. (B) A luciferase reporter assay was used to measure the effect of SolB on NRF2 activation.

Techniques Used: Activation Assay, MTT Assay, Luciferase, Reporter Assay

70) Product Images from "Protein disulfide isomerase regulates renal AT1 receptor function and blood pressure in rats"

Article Title: Protein disulfide isomerase regulates renal AT1 receptor function and blood pressure in rats

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00580.2016

Bacitracin treatment decreases Nrf2 activity in renal tissues. Nuclear Nrf2 protein levels ( A ), Keap1 protein levels ( B ), and GSK3β-pY216 protein levels ( C ) were determined by Western blotting. Top : representative blots for Nrf2 ( A ), Keap1 ( B ), and GSK3β-pY216 ( C ) and protein loading controls HDAC ( A ), GAPDH ( B ), and GSK3β ( C ). Below are ratios of the densities between Nrf2 and HDAC ( A ), Keap1 and GAPDH ( B ), and GSK3β-pY216 and GSK3β. * P
Figure Legend Snippet: Bacitracin treatment decreases Nrf2 activity in renal tissues. Nuclear Nrf2 protein levels ( A ), Keap1 protein levels ( B ), and GSK3β-pY216 protein levels ( C ) were determined by Western blotting. Top : representative blots for Nrf2 ( A ), Keap1 ( B ), and GSK3β-pY216 ( C ) and protein loading controls HDAC ( A ), GAPDH ( B ), and GSK3β ( C ). Below are ratios of the densities between Nrf2 and HDAC ( A ), Keap1 and GAPDH ( B ), and GSK3β-pY216 and GSK3β. * P

Techniques Used: Activity Assay, Western Blot

71) Product Images from "p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming"

Article Title: p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming

Journal: Nature Communications

doi: 10.1038/ncomms12030

K67, a chemical compound that inhibits the interaction between Keap1 and S349-phosphorylated p62. ( a ) Working hypothesis about an inhibitors of the interaction between Keap1 and S349-phosphorylated p62. ( b ) Structural formulas of K67 and Cpd16. ( c ) Crystal structure of Keap1-DC (white) in complex with K67 (cyan). Overall structure of Keap1-DC, shown as a ribbon model. K67 (cyan) and some of the potential interacting residues of Keap1-DC are shown in stick representation. A close-up view of K67 bound to Keap1-DC is shown. Intermolecular electrostatic interactions are depicted as broken red lines. ( d ) In vitro pull-down assay. Full-length Keap1 was allowed to form a complex with K67 (left panel) or Cdp16 (right panel) at the indicated concentration. Upper panel: Keap1 binding to His-GST-tagged Neh2(Δ17-51) was estimated by Oriole fluorescent gel staining. Middle and bottom panels: Keap1 binding to OSF-p-p62 (middle panel) or OSF-Nrf2 (bottom panel) was estimated by immunoblot analysis with anti-Keap1 antibody. Data are representative of three independent experiments. ( e ) Modelling of Keap1 (white)–K67 (cyan)–DLGex (green; upper left panel) and Keap1–Cpd16 (orange)–DLGex (lower left panel). Upper right: schematic representation of the interface between DLGex and K67. Lower right: schematic representation of the interface between DLGex and Cpd16. Potential hydrogen bonds are indicated by dashed lines between the atoms involved, whereas hydrophobic contacts are represented by an arc with spokes radiating towards the ligand atoms they contact. ( f ) In vitro pull-down assay. Full-length Keap1 was allowed to form a complex with K67 (left panel) or Cdp16 (right panel) at the indicated concentration. Keap1 binding to His-GST-tagged Neh2(1-56) was estimated by Oriole fluorescent gel staining. Data are representative of three independent experiments. ( g ) In vitro pull-down assay with biotinylated compounds. His-GST-tagged Neh2(1-56) binding to the Keap1-DC complexed with biotinylated K67 or Cpd16 was estimated by Coomassie brilliant blue staining. Data are representative of three independent experiments.
Figure Legend Snippet: K67, a chemical compound that inhibits the interaction between Keap1 and S349-phosphorylated p62. ( a ) Working hypothesis about an inhibitors of the interaction between Keap1 and S349-phosphorylated p62. ( b ) Structural formulas of K67 and Cpd16. ( c ) Crystal structure of Keap1-DC (white) in complex with K67 (cyan). Overall structure of Keap1-DC, shown as a ribbon model. K67 (cyan) and some of the potential interacting residues of Keap1-DC are shown in stick representation. A close-up view of K67 bound to Keap1-DC is shown. Intermolecular electrostatic interactions are depicted as broken red lines. ( d ) In vitro pull-down assay. Full-length Keap1 was allowed to form a complex with K67 (left panel) or Cdp16 (right panel) at the indicated concentration. Upper panel: Keap1 binding to His-GST-tagged Neh2(Δ17-51) was estimated by Oriole fluorescent gel staining. Middle and bottom panels: Keap1 binding to OSF-p-p62 (middle panel) or OSF-Nrf2 (bottom panel) was estimated by immunoblot analysis with anti-Keap1 antibody. Data are representative of three independent experiments. ( e ) Modelling of Keap1 (white)–K67 (cyan)–DLGex (green; upper left panel) and Keap1–Cpd16 (orange)–DLGex (lower left panel). Upper right: schematic representation of the interface between DLGex and K67. Lower right: schematic representation of the interface between DLGex and Cpd16. Potential hydrogen bonds are indicated by dashed lines between the atoms involved, whereas hydrophobic contacts are represented by an arc with spokes radiating towards the ligand atoms they contact. ( f ) In vitro pull-down assay. Full-length Keap1 was allowed to form a complex with K67 (left panel) or Cdp16 (right panel) at the indicated concentration. Keap1 binding to His-GST-tagged Neh2(1-56) was estimated by Oriole fluorescent gel staining. Data are representative of three independent experiments. ( g ) In vitro pull-down assay with biotinylated compounds. His-GST-tagged Neh2(1-56) binding to the Keap1-DC complexed with biotinylated K67 or Cpd16 was estimated by Coomassie brilliant blue staining. Data are representative of three independent experiments.

Techniques Used: In Vitro, Pull Down Assay, Concentration Assay, Binding Assay, Staining

Gene expression of Nrf2-targets in HCC cells harbouring phospho-mimetic p62. Quantification of mRNA levels of Nrf2 target genes in the indicated HCC cells expressing GFP, FLAG-p62 or its mutants. Values were normalized to the amount of mRNA in HCC cells expressing GFP. The experiments were performed three times. Data are presented as means±s.e. * P
Figure Legend Snippet: Gene expression of Nrf2-targets in HCC cells harbouring phospho-mimetic p62. Quantification of mRNA levels of Nrf2 target genes in the indicated HCC cells expressing GFP, FLAG-p62 or its mutants. Values were normalized to the amount of mRNA in HCC cells expressing GFP. The experiments were performed three times. Data are presented as means±s.e. * P

Techniques Used: Expressing

Persistent activation of Nrf2 in autophagy-deficient mouse livers. ( a ) Immunoblot analysis. Liver homogenates and nuclear fractions of female Atg7 f/f , Atg7 f/f ;Alb- Cre , Atg7 f/f ; Nrf2 f/f and Atg7 f/f ; Nrf2 f/f ;Alb- Cre mice aged at 5 weeks were prepared, and were subjected to immunoblotting with the indicated antibodies. ( b ) Immunofluorescence analysis. Liver sections described in a were double-immunostained with a combination of anti-phosphorylated p62 (green) and anti-p62 (red) antibodies, or of anti-Keap1 (green) and anti-p62 (red) antibodies. Scale bars, 20 μm. ( c ) Nrf2-dependent gene expressions in autophagy-deficient livers. Total RNAs were prepared from livers of female Atg7 f/f (n=4), Atg7 f/f ;Alb- Cre (n=4), Atg7 f/f ; Nrf2 f/f ( n =4) and Atg7 f/f ; Nrf2 f/f ;Alb- Cre mice ( n =4) aged at 5 weeks. Values were normalized to the amount of mRNA in the livers of Atg7 f/f or Atg7 f/f ; Nrf2 f/f mice. The experiments were performed three times. Data are means±s.e. * P
Figure Legend Snippet: Persistent activation of Nrf2 in autophagy-deficient mouse livers. ( a ) Immunoblot analysis. Liver homogenates and nuclear fractions of female Atg7 f/f , Atg7 f/f ;Alb- Cre , Atg7 f/f ; Nrf2 f/f and Atg7 f/f ; Nrf2 f/f ;Alb- Cre mice aged at 5 weeks were prepared, and were subjected to immunoblotting with the indicated antibodies. ( b ) Immunofluorescence analysis. Liver sections described in a were double-immunostained with a combination of anti-phosphorylated p62 (green) and anti-p62 (red) antibodies, or of anti-Keap1 (green) and anti-p62 (red) antibodies. Scale bars, 20 μm. ( c ) Nrf2-dependent gene expressions in autophagy-deficient livers. Total RNAs were prepared from livers of female Atg7 f/f (n=4), Atg7 f/f ;Alb- Cre (n=4), Atg7 f/f ; Nrf2 f/f ( n =4) and Atg7 f/f ; Nrf2 f/f ;Alb- Cre mice ( n =4) aged at 5 weeks. Values were normalized to the amount of mRNA in the livers of Atg7 f/f or Atg7 f/f ; Nrf2 f/f mice. The experiments were performed three times. Data are means±s.e. * P

Techniques Used: Activation Assay, Mouse Assay, Immunofluorescence

Activation of Nrf2 in HCC cells expressing phospho-mimetic p62. ( a ) Enzymes involved in glucose and glutamine metabolism regulated by Nrf2. ( b ) Immunoblot analysis. FLAG-tagged p62 and its mutants were overproduced in the indicated HCC cells using an adenovirus vector. At 48 h after infection, cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the specified antibodies. Data were obtained from three independent experiments.
Figure Legend Snippet: Activation of Nrf2 in HCC cells expressing phospho-mimetic p62. ( a ) Enzymes involved in glucose and glutamine metabolism regulated by Nrf2. ( b ) Immunoblot analysis. FLAG-tagged p62 and its mutants were overproduced in the indicated HCC cells using an adenovirus vector. At 48 h after infection, cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the specified antibodies. Data were obtained from three independent experiments.

Techniques Used: Activation Assay, Expressing, Plasmid Preparation, Infection

Inhibitory effects of K67 on tumour growth and tolerance to anti-cancer agents. ( a ) Immunoprecipitation assays. Huh1 cells expressing FLAG-Keap1 were cultured in the presence or absence of indicated compound (50 μM) for 12 h. The immunoprecipitant with anti-FLAG antibody was subjected to immunoblot analysis. Experiments were performed three times. ( b ) Ubiquitination of Nrf2. Huh7 cells expressing S351E were cultured in the presence or absence of indicated compound (50 μM). After 4 h of treatment, the cells were treated with or without lactacystin (10 μM) for 8 h. The immunoprecipitant with anti-Nrf2 antibody was subjected to immunoblot analysis. Experiments were performed three times. ( c ) Immunoblot analysis. Huh1 cells were cultured in the presence of 50 μM K67 or Cpd16 for the indicated times. Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis. Experiments were performed three times. ( d ) Quantification of mRNA levels of Nrf2 target genes in Huh1 cells treated with 50 μM K67 for the indicated times. Values were normalized to the amount of mRNA in non-treated Huh1 cells. Experiments were performed three times. Data represent means±s.e. * P
Figure Legend Snippet: Inhibitory effects of K67 on tumour growth and tolerance to anti-cancer agents. ( a ) Immunoprecipitation assays. Huh1 cells expressing FLAG-Keap1 were cultured in the presence or absence of indicated compound (50 μM) for 12 h. The immunoprecipitant with anti-FLAG antibody was subjected to immunoblot analysis. Experiments were performed three times. ( b ) Ubiquitination of Nrf2. Huh7 cells expressing S351E were cultured in the presence or absence of indicated compound (50 μM). After 4 h of treatment, the cells were treated with or without lactacystin (10 μM) for 8 h. The immunoprecipitant with anti-Nrf2 antibody was subjected to immunoblot analysis. Experiments were performed three times. ( c ) Immunoblot analysis. Huh1 cells were cultured in the presence of 50 μM K67 or Cpd16 for the indicated times. Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis. Experiments were performed three times. ( d ) Quantification of mRNA levels of Nrf2 target genes in Huh1 cells treated with 50 μM K67 for the indicated times. Values were normalized to the amount of mRNA in non-treated Huh1 cells. Experiments were performed three times. Data represent means±s.e. * P

Techniques Used: Immunoprecipitation, Expressing, Cell Culture

Metabolic intermediates in autophagy-deficient mouse livers. Quantification of metabolic intermediates in glucose and glutamine metabolism in livers of female Atg7 f/f ( n =5), Atg7 f/f ;Alb- Cre ( n =5), Atg7 f/f ; Nrf2 f/f ( n =10) and Atg7 f/f ; Nrf2 f/f ;Alb- Cre mice ( n =13) aged at 5 weeks. The amount of glycogen was also quantified. Data are means±s.e. * P
Figure Legend Snippet: Metabolic intermediates in autophagy-deficient mouse livers. Quantification of metabolic intermediates in glucose and glutamine metabolism in livers of female Atg7 f/f ( n =5), Atg7 f/f ;Alb- Cre ( n =5), Atg7 f/f ; Nrf2 f/f ( n =10) and Atg7 f/f ; Nrf2 f/f ;Alb- Cre mice ( n =13) aged at 5 weeks. The amount of glycogen was also quantified. Data are means±s.e. * P

Techniques Used: Mouse Assay

72) Product Images from "Sulforaphane Protects the Liver against CdSe Quantum Dot-Induced Cytotoxicity"

Article Title: Sulforaphane Protects the Liver against CdSe Quantum Dot-Induced Cytotoxicity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0138771

Effect of knockdown TR-1, Keap1 and Nrf2 on cytotoxicity in HHL-5 cells exposed to CdSe QDs. TR-1, Keap1 or Nrf2 were knocked down, respectively. Allstars (AS) were used as a negative control. Cells were incubated with 5 μM SFN or DMSO (0.1%) control for 24 h then exposed to 20 μM CdSe QDs for 24 h. Cell viability was measured by MTT assay, and data shown as means ± SD (n = 6). Significant levels (*P
Figure Legend Snippet: Effect of knockdown TR-1, Keap1 and Nrf2 on cytotoxicity in HHL-5 cells exposed to CdSe QDs. TR-1, Keap1 or Nrf2 were knocked down, respectively. Allstars (AS) were used as a negative control. Cells were incubated with 5 μM SFN or DMSO (0.1%) control for 24 h then exposed to 20 μM CdSe QDs for 24 h. Cell viability was measured by MTT assay, and data shown as means ± SD (n = 6). Significant levels (*P

Techniques Used: Negative Control, Incubation, MTT Assay

73) Product Images from "Docosahexaenoic Acid Induces Expression of Heme Oxygenase-1 and NAD(P)H:quinone Oxidoreductase through Activation of Nrf2 in Human Mammary Epithelial Cells"

Article Title: Docosahexaenoic Acid Induces Expression of Heme Oxygenase-1 and NAD(P)H:quinone Oxidoreductase through Activation of Nrf2 in Human Mammary Epithelial Cells

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules22060969

Possible involvement of Keap1 cysteine thiol modification in DHA-induced HO-1 and NQO1 expression. ( A ) MCF-10A cells were pretreated with or without a thiol reducing agent DTT (500 μM) for 1 h, followed by 18-h incubation with 25 μM DHA. ( B ) Cells were pretreated with another thiol reducing agent NEM (25 μM) for 1 h, followed by 18-h incubation with 25 μM DHA. ( C ) MCF-10A cells were transfected with HA-mock, HA-Keap1 WT, Keap1-C151S, Keap1-C273S, or Keap1-C288S expressing vector for 24 h, and incubated with DHA (25 μM) for another 9 h to determine the expression of HO-1 and NQO1. HA-Keap1 was used to ensure the equal expression of mutant vectors. Each blot is a representative of three different experiments. Columns, means (n = 3); bars, SD. *, p
Figure Legend Snippet: Possible involvement of Keap1 cysteine thiol modification in DHA-induced HO-1 and NQO1 expression. ( A ) MCF-10A cells were pretreated with or without a thiol reducing agent DTT (500 μM) for 1 h, followed by 18-h incubation with 25 μM DHA. ( B ) Cells were pretreated with another thiol reducing agent NEM (25 μM) for 1 h, followed by 18-h incubation with 25 μM DHA. ( C ) MCF-10A cells were transfected with HA-mock, HA-Keap1 WT, Keap1-C151S, Keap1-C273S, or Keap1-C288S expressing vector for 24 h, and incubated with DHA (25 μM) for another 9 h to determine the expression of HO-1 and NQO1. HA-Keap1 was used to ensure the equal expression of mutant vectors. Each blot is a representative of three different experiments. Columns, means (n = 3); bars, SD. *, p

Techniques Used: Modification, Expressing, Incubation, Transfection, Plasmid Preparation, Mutagenesis

74) Product Images from "Oxidative stress and cell damage in a model of precancerous lesions and advanced hepatocellular carcinoma in rats"

Article Title: Oxidative stress and cell damage in a model of precancerous lesions and advanced hepatocellular carcinoma in rats

Journal: Toxicology Reports

doi: 10.1016/j.toxrep.2014.11.015

Western blot analysis of TGF-1β, eNOS, and iNOS. Protein from liver extracts was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. (A) Representative images. (B) Densitometric quantification. Values are expressed as means ± SD ( n = 5). a p
Figure Legend Snippet: Western blot analysis of TGF-1β, eNOS, and iNOS. Protein from liver extracts was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. (A) Representative images. (B) Densitometric quantification. Values are expressed as means ± SD ( n = 5). a p

Techniques Used: Western Blot, Polyacrylamide Gel Electrophoresis

75) Product Images from "Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels"

Article Title: Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels

Journal: Scientific Reports

doi: 10.1038/s41598-018-26049-5

Effect of plasma on chicken sperm mRNA and protein expression. Semen of 60-week-old cocks was exposed to varying plasma potentials for 20 s. Relative mRNA levels of the following genes were measured: ( a ) NOX4 , NRF2 , and KEAP1 ; ( b ) SOD , CAT , and GPx ; ( c ) PRDX1 , PRDX 3, PRDX4 , and PRDX6 ; ( d ) ATP5A1 , ATP5B , ATP5C1 , ATP5F1 , ATP5G1 , ATP5G3 , ATP5H , ATP5I , ATP5J , ATP5J 2, ATP5L , and ATP5S ; and ( e ) AMPKα2 , AMPKβ2 , AMPKγ3 , and mTOR . ( f ) Western blot analysis of protein bands. Uncropped immunoblot scans are presented in Supplementary Figure S1 . The grouping of gels/blots cropped from different gels. All blots were visualized with 5 min exposure time. Relative protein levels of ( g ) NRF2, KEAP1, PRDX4, ( h ) ATP5A, ( i ) p-AMPKα/AMPKα, and ( j ) p-mTOR/mTOR. Values are expressed as the mean ± standard error (n = 3) of three replicates; n represents an individual cock. * p
Figure Legend Snippet: Effect of plasma on chicken sperm mRNA and protein expression. Semen of 60-week-old cocks was exposed to varying plasma potentials for 20 s. Relative mRNA levels of the following genes were measured: ( a ) NOX4 , NRF2 , and KEAP1 ; ( b ) SOD , CAT , and GPx ; ( c ) PRDX1 , PRDX 3, PRDX4 , and PRDX6 ; ( d ) ATP5A1 , ATP5B , ATP5C1 , ATP5F1 , ATP5G1 , ATP5G3 , ATP5H , ATP5I , ATP5J , ATP5J 2, ATP5L , and ATP5S ; and ( e ) AMPKα2 , AMPKβ2 , AMPKγ3 , and mTOR . ( f ) Western blot analysis of protein bands. Uncropped immunoblot scans are presented in Supplementary Figure S1 . The grouping of gels/blots cropped from different gels. All blots were visualized with 5 min exposure time. Relative protein levels of ( g ) NRF2, KEAP1, PRDX4, ( h ) ATP5A, ( i ) p-AMPKα/AMPKα, and ( j ) p-mTOR/mTOR. Values are expressed as the mean ± standard error (n = 3) of three replicates; n represents an individual cock. * p

Techniques Used: Expressing, Western Blot

Cytosine methylation analysis of chicken spermatozoa. Cytosine methylation of ( a ) NRF 2, ( b ) KEAP1 , ( c ) PRDX4 , ( d ) ATP5A1 , ( e ) AMPKα2 , and ( f ) mTOR were analyzed using CyMATE. Lengths of sequenced regions and position of cytosine are shown schematically. The order of the individual sequences of ten clones is listed on the left. 0 represents the control group; 11.7 represents plasma-treated group at 11.7 kV for 20 s; and 27.6 represents plasma-treated group at 27.6 kV for 20 s. The reference sequence is shown in the first line. The sequence is distinguished by circles for CG, squares for CHG, and triangles for CHH. Filled symbols represent methylated cytosine, and open symbols represent unmethylated cytosine. Average methylation levels for CG, CHG, and CHH of ( g ) NRF2 , ( h ) KEAP1 , ( i ) PRDX4 , ( j ) ATP5A1 , ( k ) AMPKα2 , and ( l ) mTOR .
Figure Legend Snippet: Cytosine methylation analysis of chicken spermatozoa. Cytosine methylation of ( a ) NRF 2, ( b ) KEAP1 , ( c ) PRDX4 , ( d ) ATP5A1 , ( e ) AMPKα2 , and ( f ) mTOR were analyzed using CyMATE. Lengths of sequenced regions and position of cytosine are shown schematically. The order of the individual sequences of ten clones is listed on the left. 0 represents the control group; 11.7 represents plasma-treated group at 11.7 kV for 20 s; and 27.6 represents plasma-treated group at 27.6 kV for 20 s. The reference sequence is shown in the first line. The sequence is distinguished by circles for CG, squares for CHG, and triangles for CHH. Filled symbols represent methylated cytosine, and open symbols represent unmethylated cytosine. Average methylation levels for CG, CHG, and CHH of ( g ) NRF2 , ( h ) KEAP1 , ( i ) PRDX4 , ( j ) ATP5A1 , ( k ) AMPKα2 , and ( l ) mTOR .

Techniques Used: Methylation, Clone Assay, Sequencing

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 ( ). ..

MTT Assay:

Article Title: Anti-Inflammatory Activities of Leaf Oil from Cinnamomum subavenium In Vitro and In Vivo
Article Snippet: Chemicals and Materials Indo, LPS (Escherichia coli serotype 0111:B4), λ -carrageenan, and MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) were obtained from Sigma-Aldrich Co. (USA). .. Anti-iNOS, anti-COX-2 antibody, and anti-NF-κ B p65 antibody were from Santa Cruz Biotechnology (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.

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.

Enzyme-linked Immunosorbent Assay:

Article Title: Anti-Inflammatory Activities of Leaf Oil from Cinnamomum subavenium In Vitro and In Vivo
Article Snippet: TNF-α , IL-1β , and IL-6 ELISA were from Boster Biological Engineering Co., Ltd. (China). .. Anti-iNOS, anti-COX-2 antibody, and anti-NF-κ B p65 antibody were from Santa Cruz Biotechnology (USA).

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: 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 ( ).

Modification:

Article Title: Steppogenin Isolated from Cudrania tricuspidata Shows Antineuroinflammatory Effects via NF-κB and MAPK Pathways in LPS-Stimulated BV2 and Primary Rat Microglial Cells
Article Snippet: Chemicals and Reagents Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL Co. (Grand Island, NY, USA). .. Primary antibodies, including mouse/goat/rabbit anti-COX-2 (sc-1745), anti-iNOS (sc-650), anti-β-actin (sc-47778), anti-IкB-α (sc-371), anti-phospho-IкB-α (sc-8404), anti-p50 (sc-7178), anti-p65 (sc-8008), and anti-proliferating cell nuclear antigen (PCNA) (sc-7907), and secondary antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

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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  • 99
    Santa Cruz Biotechnology anti keap1
    Dose response of Nrf2 transcriptional activation by cinnamic aldehyde and an ethanolic cinnamon extract in MDA-MB231 breast carcinoma and HCT116 colon carcinoma cells. (a–b) MDA-MB-231 cells (panel a) or HCT116 (panel b) were cotransfected with plasmids containing a GST-ARE-firefly luciferase reporter gene and expression plasmids for the Nrf2 and <t>Keap1</t> proteins. A plasmid encoding renilla luciferase, driven by the herpes simplex virus thymidine kinase promoter, was included in all transfections to normalize transfection efficiency. Twenty-four hours post-transfection, cells were dosed with the indicated concentrations of each compound (tBHQ: 50μM) for 16h. Firefly and renilla luciferase activity was measured and is expressed as relative activity (F/R) compared to untreated control ( p
    Anti Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Santa Cruz Biotechnology polyclonal primary antibody against keap1
    Dose response of Nrf2 transcriptional activation by cinnamic aldehyde and an ethanolic cinnamon extract in MDA-MB231 breast carcinoma and HCT116 colon carcinoma cells. (a–b) MDA-MB-231 cells (panel a) or HCT116 (panel b) were cotransfected with plasmids containing a GST-ARE-firefly luciferase reporter gene and expression plasmids for the Nrf2 and <t>Keap1</t> proteins. A plasmid encoding renilla luciferase, driven by the herpes simplex virus thymidine kinase promoter, was included in all transfections to normalize transfection efficiency. Twenty-four hours post-transfection, cells were dosed with the indicated concentrations of each compound (tBHQ: 50μM) for 16h. Firefly and renilla luciferase activity was measured and is expressed as relative activity (F/R) compared to untreated control ( p
    Polyclonal Primary Antibody Against Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 81/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology goat anti keap1 polyclonal antibody e20
    Dose response of Nrf2 transcriptional activation by cinnamic aldehyde and an ethanolic cinnamon extract in MDA-MB231 breast carcinoma and HCT116 colon carcinoma cells. (a–b) MDA-MB-231 cells (panel a) or HCT116 (panel b) were cotransfected with plasmids containing a GST-ARE-firefly luciferase reporter gene and expression plasmids for the Nrf2 and <t>Keap1</t> proteins. A plasmid encoding renilla luciferase, driven by the herpes simplex virus thymidine kinase promoter, was included in all transfections to normalize transfection efficiency. Twenty-four hours post-transfection, cells were dosed with the indicated concentrations of each compound (tBHQ: 50μM) for 16h. Firefly and renilla luciferase activity was measured and is expressed as relative activity (F/R) compared to untreated control ( p
    Goat Anti Keap1 Polyclonal Antibody E20, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Dose response of Nrf2 transcriptional activation by cinnamic aldehyde and an ethanolic cinnamon extract in MDA-MB231 breast carcinoma and HCT116 colon carcinoma cells. (a–b) MDA-MB-231 cells (panel a) or HCT116 (panel b) were cotransfected with plasmids containing a GST-ARE-firefly luciferase reporter gene and expression plasmids for the Nrf2 and Keap1 proteins. A plasmid encoding renilla luciferase, driven by the herpes simplex virus thymidine kinase promoter, was included in all transfections to normalize transfection efficiency. Twenty-four hours post-transfection, cells were dosed with the indicated concentrations of each compound (tBHQ: 50μM) for 16h. Firefly and renilla luciferase activity was measured and is expressed as relative activity (F/R) compared to untreated control ( p

    Journal: Molecules (Basel, Switzerland)

    Article Title: The Cinnamon-derived Dietary Factor Cinnamic Aldehyde Activates the Nrf2-dependent Antioxidant Response in Human Epithelial Colon Cells

    doi: 10.3390/molecules15053338

    Figure Lengend Snippet: Dose response of Nrf2 transcriptional activation by cinnamic aldehyde and an ethanolic cinnamon extract in MDA-MB231 breast carcinoma and HCT116 colon carcinoma cells. (a–b) MDA-MB-231 cells (panel a) or HCT116 (panel b) were cotransfected with plasmids containing a GST-ARE-firefly luciferase reporter gene and expression plasmids for the Nrf2 and Keap1 proteins. A plasmid encoding renilla luciferase, driven by the herpes simplex virus thymidine kinase promoter, was included in all transfections to normalize transfection efficiency. Twenty-four hours post-transfection, cells were dosed with the indicated concentrations of each compound (tBHQ: 50μM) for 16h. Firefly and renilla luciferase activity was measured and is expressed as relative activity (F/R) compared to untreated control ( p

    Article Snippet: Western blot analysis of Nrf2, Keap1, γ-GCS and HO-1 was performed as published recently using anti-Nrf2 (H-300, rabbit polyclonal), anti-Keap1 (E-20, goat polyclonal) anti-GCSm (E-4, mouse monoclonal), and anti-heme oxygenase 1 (H-105, rabbit polyclonal) antibodies, respectively (Santa Cruz Biotechnology, Santa Cruz, CA) [ – ].

    Techniques: Activation Assay, Multiple Displacement Amplification, Luciferase, Expressing, Plasmid Preparation, Transfection, Activity Assay