Structured Review

Santa Cruz Biotechnology anti nrf2
Proposed model of interaction of miR-433 with regulators and targets. miR-433 is susceptible to regulation by ROS and TGF-β. ROS induces activation of the <t>Nrf2</t> pathway, an important mediator in the expression of GCLs. On the other hand, ROS and TGF-β are reciprocally regulated. An increase in the activation in TGF-β results in elevated miR-433 levels targeting GCLc and GCLm, contributing to both reduced GSH levels and nucleophilic tone. ROS, reactive oxygen species.
Anti Nrf2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti nrf2/product/Santa Cruz Biotechnology
Average 99 stars, based on 152 article reviews
Price from $9.99 to $1999.99
anti nrf2 - by Bioz Stars, 2020-02
99/100 stars

Images

1) Product Images from "Targeting of Gamma-Glutamyl-Cysteine Ligase by miR-433 Reduces Glutathione Biosynthesis and Promotes TGF-β-Dependent Fibrogenesis"

Article Title: Targeting of Gamma-Glutamyl-Cysteine Ligase by miR-433 Reduces Glutathione Biosynthesis and Promotes TGF-β-Dependent Fibrogenesis

Journal: Antioxidants & Redox Signaling

doi: 10.1089/ars.2014.6025

Proposed model of interaction of miR-433 with regulators and targets. miR-433 is susceptible to regulation by ROS and TGF-β. ROS induces activation of the Nrf2 pathway, an important mediator in the expression of GCLs. On the other hand, ROS and TGF-β are reciprocally regulated. An increase in the activation in TGF-β results in elevated miR-433 levels targeting GCLc and GCLm, contributing to both reduced GSH levels and nucleophilic tone. ROS, reactive oxygen species.
Figure Legend Snippet: Proposed model of interaction of miR-433 with regulators and targets. miR-433 is susceptible to regulation by ROS and TGF-β. ROS induces activation of the Nrf2 pathway, an important mediator in the expression of GCLs. On the other hand, ROS and TGF-β are reciprocally regulated. An increase in the activation in TGF-β results in elevated miR-433 levels targeting GCLc and GCLm, contributing to both reduced GSH levels and nucleophilic tone. ROS, reactive oxygen species.

Techniques Used: Activation Assay, Expressing

miR-433 does not regulate Nrf2, but Nrf2 is necessary for the full expression of GCLs and miR-433. (A) Shown on top is a Western blot depicting Nrf2 protein levels in HUVEC transfected with a negative control (miR-NC), miR-144, or miR-433 (all 40 n M , 48 h). Bar graphs represent mean±SEM of n ≥3 experiments for protein ( left ) and mRNA levels ( right ). (B) Effect of Nrf2 RNA silencing on GCLc and GCLm expression (protein and mRNA levels) in HUVEC. Representative Western blot ( top left ) of Nrf2 and GCLs protein levels after treatment with siRNA control (SC) or siRNA for Nrf2 (40 n M , 48 h). Bar graphs represent GCLs mRNA ( top right panel ) and miR-433 ( lower panel ) levels after Nrf2 silencing, mean±SEM of at least three experiments, * p
Figure Legend Snippet: miR-433 does not regulate Nrf2, but Nrf2 is necessary for the full expression of GCLs and miR-433. (A) Shown on top is a Western blot depicting Nrf2 protein levels in HUVEC transfected with a negative control (miR-NC), miR-144, or miR-433 (all 40 n M , 48 h). Bar graphs represent mean±SEM of n ≥3 experiments for protein ( left ) and mRNA levels ( right ). (B) Effect of Nrf2 RNA silencing on GCLc and GCLm expression (protein and mRNA levels) in HUVEC. Representative Western blot ( top left ) of Nrf2 and GCLs protein levels after treatment with siRNA control (SC) or siRNA for Nrf2 (40 n M , 48 h). Bar graphs represent GCLs mRNA ( top right panel ) and miR-433 ( lower panel ) levels after Nrf2 silencing, mean±SEM of at least three experiments, * p

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

2) Product Images from "Phloretin Attenuates Allergic Airway Inflammation and Oxidative Stress in Asthmatic Mice"

Article Title: Phloretin Attenuates Allergic Airway Inflammation and Oxidative Stress in Asthmatic Mice

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00134

Phloretin (PT) effects on oxidative stress factors . (A) Western blot shows PT modulation of HO-1 and Nrf2 expression in lung tissue of normal (N) and OVA-stimulated (OVA) mice, without or with PT (PT5-20) treatment. (B) Malondialdehyde (MDA) activity and (C) GSH activity in lung tissues of mice. Data are presented as the mean ± SEM. * p
Figure Legend Snippet: Phloretin (PT) effects on oxidative stress factors . (A) Western blot shows PT modulation of HO-1 and Nrf2 expression in lung tissue of normal (N) and OVA-stimulated (OVA) mice, without or with PT (PT5-20) treatment. (B) Malondialdehyde (MDA) activity and (C) GSH activity in lung tissues of mice. Data are presented as the mean ± SEM. * p

Techniques Used: Western Blot, Expressing, Mouse Assay, Multiple Displacement Amplification, Activity Assay

3) Product Images from "Sinomenine Regulates Inflammatory Response and Oxidative Stress via Nuclear Factor kappa B (NF-κB) and NF-E2-Related Factor 2 (Nrf2) Signaling Pathways in Ankle Fractures in Children"

Article Title: Sinomenine Regulates Inflammatory Response and Oxidative Stress via Nuclear Factor kappa B (NF-κB) and NF-E2-Related Factor 2 (Nrf2) Signaling Pathways in Ankle Fractures in Children

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.910740

Effect of SIN on oxidative stress. After treatment with 0.25, 0.5, or 1 mM for 24 h, the level of MDA ( A ) and activity of SOD ( B ) and CAT ( C ) was detected; Protein ( D ) and mRNA ( E ) levels of Nrf2, HO-1, and NQO-1 were detected using Western blotting and qRT-PCR, respectively. Controls: cells without any treatment; BK: 1 μg/ml BK treatment group; BK + SIN 0.25: 1 μg/ml BK + 0.25 nM SIN treatment group; BK + SIN 0.5: 1 μg/ml BK + 0.5 nM SIN treatment group; BK + SIN 1: 1 μg/ml BK + 1 nM SIN treatment group; * p
Figure Legend Snippet: Effect of SIN on oxidative stress. After treatment with 0.25, 0.5, or 1 mM for 24 h, the level of MDA ( A ) and activity of SOD ( B ) and CAT ( C ) was detected; Protein ( D ) and mRNA ( E ) levels of Nrf2, HO-1, and NQO-1 were detected using Western blotting and qRT-PCR, respectively. Controls: cells without any treatment; BK: 1 μg/ml BK treatment group; BK + SIN 0.25: 1 μg/ml BK + 0.25 nM SIN treatment group; BK + SIN 0.5: 1 μg/ml BK + 0.5 nM SIN treatment group; BK + SIN 1: 1 μg/ml BK + 1 nM SIN treatment group; * p

Techniques Used: Multiple Displacement Amplification, Activity Assay, Western Blot, Quantitative RT-PCR

4) Product Images from "C66 ameliorates diabetic nephropathy in mice by both upregulating NRF2 function via increase in miR-200a and inhibiting miR-21"

Article Title: C66 ameliorates diabetic nephropathy in mice by both upregulating NRF2 function via increase in miR-200a and inhibiting miR-21

Journal: Diabetologia

doi: 10.1007/s00125-016-3958-8

Possible mechanisms for the prevention of diabetic nephropathy by C66. On one hand, C66 upregulates miR-200a to enhance NRF2 function by targeting Keap1 , leading to alleviation of renal oxidative damage. On the other hand, C66 inhibits miR-21-induced
Figure Legend Snippet: Possible mechanisms for the prevention of diabetic nephropathy by C66. On one hand, C66 upregulates miR-200a to enhance NRF2 function by targeting Keap1 , leading to alleviation of renal oxidative damage. On the other hand, C66 inhibits miR-21-induced

Techniques Used:

Both C66 and LNA-21 downregulate miR-21 in Nrf2 -null mice, resulting in reduced levels of stimulators for diabetic renal fibrosis. ( a , b ) In Nrf2 -null mice, expression levels of renal miR-21 ( a ) and Smad7 ( b ) RNA were evaluated by RT-PCR. ( c –
Figure Legend Snippet: Both C66 and LNA-21 downregulate miR-21 in Nrf2 -null mice, resulting in reduced levels of stimulators for diabetic renal fibrosis. ( a , b ) In Nrf2 -null mice, expression levels of renal miR-21 ( a ) and Smad7 ( b ) RNA were evaluated by RT-PCR. ( c –

Techniques Used: Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Deletion of the Nrf2 gene partially abolished the renal protection afforded by C66 against diabetes-induced albuminuria. ( a , b ) Blood glucose levels in C57 WT mice ( a ) and Nrf2 -null mice ( b ) were monitored at 0, 4, 8, 12, 16, 20 and 24 weeks after diabetes
Figure Legend Snippet: Deletion of the Nrf2 gene partially abolished the renal protection afforded by C66 against diabetes-induced albuminuria. ( a , b ) Blood glucose levels in C57 WT mice ( a ) and Nrf2 -null mice ( b ) were monitored at 0, 4, 8, 12, 16, 20 and 24 weeks after diabetes

Techniques Used: Mouse Assay

C66 upregulation of renal NRF2 requires an increase in miR-200a. ( a – e ) The effects of C66, diabetes, LNA-200a and their combinations were compared on RNA levels of miR-200a ( a ) and Keap1 ( b ) and on protein levels of KEAP1 ( c ), total NRF2 ( d ) and
Figure Legend Snippet: C66 upregulation of renal NRF2 requires an increase in miR-200a. ( a – e ) The effects of C66, diabetes, LNA-200a and their combinations were compared on RNA levels of miR-200a ( a ) and Keap1 ( b ) and on protein levels of KEAP1 ( c ), total NRF2 ( d ) and

Techniques Used:

Both C66 and LNA-21 attenuated diabetes-induced pathological changes and albuminuria in the absence of NRF2. ( a – d ) In Nrf2 -null mice, PAS and Masson’s trichrome staining ( a ) were performed with glomerular area ( b ) and mesangial matrix
Figure Legend Snippet: Both C66 and LNA-21 attenuated diabetes-induced pathological changes and albuminuria in the absence of NRF2. ( a – d ) In Nrf2 -null mice, PAS and Masson’s trichrome staining ( a ) were performed with glomerular area ( b ) and mesangial matrix

Techniques Used: Mouse Assay, Staining

5) Product Images from "Extraction, identification, and antioxidant property evaluation of limonin from pummelo seeds"

Article Title: Extraction, identification, and antioxidant property evaluation of limonin from pummelo seeds

Journal: Animal Nutrition

doi: 10.1016/j.aninu.2018.05.005

Effect of limonin on the transcriptional (A, gene expressions) and posttranscriptional of Nrf2-ARE pathway (B, protein synthesis). Significance is marked by star bars. * stands for P
Figure Legend Snippet: Effect of limonin on the transcriptional (A, gene expressions) and posttranscriptional of Nrf2-ARE pathway (B, protein synthesis). Significance is marked by star bars. * stands for P

Techniques Used:

6) Product Images from "Contribution of Nrf2 to Atherogenic Phenotype Switching of Coronary Arterial Smooth Muscle Cells Lacking CD38 Gene"

Article Title: Contribution of Nrf2 to Atherogenic Phenotype Switching of Coronary Arterial Smooth Muscle Cells Lacking CD38 Gene

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

doi: 10.1159/000430366

Working model in this project. In this model, Nrf2 plays an important role in the occurrence of proliferative and atherosclerotic phenotype when CD38 gene was silenced or deleted. CD38-derived enzyme product cADPR and NAADP and associated Ca 2+ signaling mechanisms are importantly implicated in the control of Nrf2 activation through NOX4-dependent O 2 ·− production.
Figure Legend Snippet: Working model in this project. In this model, Nrf2 plays an important role in the occurrence of proliferative and atherosclerotic phenotype when CD38 gene was silenced or deleted. CD38-derived enzyme product cADPR and NAADP and associated Ca 2+ signaling mechanisms are importantly implicated in the control of Nrf2 activation through NOX4-dependent O 2 ·− production.

Techniques Used: Derivative Assay, Activation Assay

NOX4-dependend O 2 −. is responsible for Nrf2 translocation into the nucleus. Representative Western blot gel documents and summarized data showing the protein expression of NOX4 (A) and NOX1 (B) in CD38 +/+ and CD38 −/− CAMs treated with 7-Ket. (C) Summarized ESR data showing the relative O 2 .− production in CD38 +/+ and CD38 −/− CAMs under control and treatment with 7-Ket. * P
Figure Legend Snippet: NOX4-dependend O 2 −. is responsible for Nrf2 translocation into the nucleus. Representative Western blot gel documents and summarized data showing the protein expression of NOX4 (A) and NOX1 (B) in CD38 +/+ and CD38 −/− CAMs treated with 7-Ket. (C) Summarized ESR data showing the relative O 2 .− production in CD38 +/+ and CD38 −/− CAMs under control and treatment with 7-Ket. * P

Techniques Used: Translocation Assay, Western Blot, Expressing, Electron Paramagnetic Resonance

CD38 deficiency inhibited the activation of Nrf2. Representative Western blot gel documents and summarized data showing the protein expression of nuclear Nrf2 in CD38 −/− and CD38 +/+ CAMs (A) or CD38 −/− CAMs transfected CD38 cDNA plasmids (B). (C) Typical representative fluorescent images for nuclear translocation of Nrf2. DAPI were used as a nuclear staining marker. (D) Summarized data showing nuclear Nrf2 activity of CAMs. * P
Figure Legend Snippet: CD38 deficiency inhibited the activation of Nrf2. Representative Western blot gel documents and summarized data showing the protein expression of nuclear Nrf2 in CD38 −/− and CD38 +/+ CAMs (A) or CD38 −/− CAMs transfected CD38 cDNA plasmids (B). (C) Typical representative fluorescent images for nuclear translocation of Nrf2. DAPI were used as a nuclear staining marker. (D) Summarized data showing nuclear Nrf2 activity of CAMs. * P

Techniques Used: Activation Assay, Western Blot, Expressing, Transfection, Translocation Assay, Staining, Marker, Activity Assay

Efficiency of Nrf2 siRNA and cDNA transfection. Representative Western blot gel documents and summarized data showing the protein expression of Nrf2 after the transfection with Nrf2 siRNA (A) and cDNA(B). * P
Figure Legend Snippet: Efficiency of Nrf2 siRNA and cDNA transfection. Representative Western blot gel documents and summarized data showing the protein expression of Nrf2 after the transfection with Nrf2 siRNA (A) and cDNA(B). * P

Techniques Used: Transfection, Western Blot, Expressing

Nrf2 deficiency changed the phenotypic marker in CAMs. CD38 +/+ CAMs were transfected with Nrf2 siRNA under the treatment with 7-Ket for 24 hours. Representative Western blot gel documents and summarized data showing the protein expression of calponin (A), SM22α (B), vimentin (C), and PCNA (D). (E) Typical representative fluorescent images for α-SMA staining. (F) Summarized data showing the proliferation rate of CAMs in CD38 +/+ and CD38 −/− CAMs. * P
Figure Legend Snippet: Nrf2 deficiency changed the phenotypic marker in CAMs. CD38 +/+ CAMs were transfected with Nrf2 siRNA under the treatment with 7-Ket for 24 hours. Representative Western blot gel documents and summarized data showing the protein expression of calponin (A), SM22α (B), vimentin (C), and PCNA (D). (E) Typical representative fluorescent images for α-SMA staining. (F) Summarized data showing the proliferation rate of CAMs in CD38 +/+ and CD38 −/− CAMs. * P

Techniques Used: Marker, Transfection, Western Blot, Expressing, Staining

cADPR or NAADP-sensitive Ca 2+ regulates O 2 −. Production. CAMs were pretreated with 8-Br-cADPR (30 μM) or NED-19 (10 μM) and then stimulated with 7-Ket for 24 hours. Representative Ca 2+ traces (A) and summarized data (B) showing the level of cytosolic Ca 2+ in CAMs. (C) Summarized ESR data showing the relative O 2 −. production in CAMs before and after treatment with 8-Br-cADPR or NED-19. (D) Typical representative fluorescent images showing nuclear translocation of Nrf2 in CAMs pretreated with 8-Br-cADPR or NED-19. (E) Summarized data showing nuclear Nrf2 activity in CAMs pretreated with 8-Br-cADPR or NED-19. * P
Figure Legend Snippet: cADPR or NAADP-sensitive Ca 2+ regulates O 2 −. Production. CAMs were pretreated with 8-Br-cADPR (30 μM) or NED-19 (10 μM) and then stimulated with 7-Ket for 24 hours. Representative Ca 2+ traces (A) and summarized data (B) showing the level of cytosolic Ca 2+ in CAMs. (C) Summarized ESR data showing the relative O 2 −. production in CAMs before and after treatment with 8-Br-cADPR or NED-19. (D) Typical representative fluorescent images showing nuclear translocation of Nrf2 in CAMs pretreated with 8-Br-cADPR or NED-19. (E) Summarized data showing nuclear Nrf2 activity in CAMs pretreated with 8-Br-cADPR or NED-19. * P

Techniques Used: Electron Paramagnetic Resonance, Translocation Assay, Activity Assay

Nrf2 deficiency changed the phenotypic marker in CAMs. CD38 −/− CAMs were transfected with Nrf2 cDNA plasmids under the treatment with 7-Ket for 24 hours. Representative Western blot gel documents and summarized data showing the protein expression of calponin (A), SM22α (B), Vimentin (C), and PCNA (D). (E) Typical representative fluorescent images for α-SMA staining. (F) Summarized data showing the proliferation rate of CAMs in CD38 −/− transfected with Nrf2 cDNA. * P
Figure Legend Snippet: Nrf2 deficiency changed the phenotypic marker in CAMs. CD38 −/− CAMs were transfected with Nrf2 cDNA plasmids under the treatment with 7-Ket for 24 hours. Representative Western blot gel documents and summarized data showing the protein expression of calponin (A), SM22α (B), Vimentin (C), and PCNA (D). (E) Typical representative fluorescent images for α-SMA staining. (F) Summarized data showing the proliferation rate of CAMs in CD38 −/− transfected with Nrf2 cDNA. * P

Techniques Used: Marker, Transfection, Western Blot, Expressing, Staining

7) Product Images from "CDDO-Me Protects Normal Lung and Breast Epithelial Cells but Not Cancer Cells from Radiation"

Article Title: CDDO-Me Protects Normal Lung and Breast Epithelial Cells but Not Cancer Cells from Radiation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0115600

CDDO-Me is a potent radiation countermeasure in bronchial and breast epithelial cells, and Nrf2 knockdown abrogates these radioprotective effects. (A) Normal breast and lung epithelia are radioprotected at multiple doses of CDDO-Me. Cells were treated with drug 18 hours before exposure to 3 Gy gamma IR, then seeded immediately into clonogenicity. Colonies grown for ∼14 days before fixation with 6% glutaraldehyde/0.5% crystal violet stain. Mean ± SEM of four experiments seeded in triplicate, *p
Figure Legend Snippet: CDDO-Me is a potent radiation countermeasure in bronchial and breast epithelial cells, and Nrf2 knockdown abrogates these radioprotective effects. (A) Normal breast and lung epithelia are radioprotected at multiple doses of CDDO-Me. Cells were treated with drug 18 hours before exposure to 3 Gy gamma IR, then seeded immediately into clonogenicity. Colonies grown for ∼14 days before fixation with 6% glutaraldehyde/0.5% crystal violet stain. Mean ± SEM of four experiments seeded in triplicate, *p

Techniques Used: Staining

CDDO-Me activates the Nrf2 antioxidant pathway in epithelial cells. (A) Nrf2 Pathway: Nrf2 is a transcription factor normally bound by its cytoplasmic repressor Keap1, which acts as a molecular oxidative sensor and marks Nrf2 for degradation. When there is an abundance of reactive species in the cells, Nrf2 accumulates in the cytoplasm, eventually undergoing various phosphorylation events to translocate to the nucleus and bind to Antioxidant Response Elements (AREs) in the genome, resulting in the transcription of multiple antioxidative and cyto-protective genes. CDDO-Me acts by facilitating the dissociation between Keap1 and Nrf2, leading to Nrf2 activation. (B) Chemical structure of CDDO-Me: Oleana-1,9(11)-dien-28-oicacid, 2-cyano-3,12-dioxo-, methyl ester (RTA-402; bardoxolone-methyl). (C, D) CDDO-Me increases expression of ARE-driven luciferase 18 hours after drug treatment in HBEC 3KT and HME1, respectively. Firefly ARE-luciferase normalized to renilla control (RLU). Mean ± SEM of 6 replicates, *p
Figure Legend Snippet: CDDO-Me activates the Nrf2 antioxidant pathway in epithelial cells. (A) Nrf2 Pathway: Nrf2 is a transcription factor normally bound by its cytoplasmic repressor Keap1, which acts as a molecular oxidative sensor and marks Nrf2 for degradation. When there is an abundance of reactive species in the cells, Nrf2 accumulates in the cytoplasm, eventually undergoing various phosphorylation events to translocate to the nucleus and bind to Antioxidant Response Elements (AREs) in the genome, resulting in the transcription of multiple antioxidative and cyto-protective genes. CDDO-Me acts by facilitating the dissociation between Keap1 and Nrf2, leading to Nrf2 activation. (B) Chemical structure of CDDO-Me: Oleana-1,9(11)-dien-28-oicacid, 2-cyano-3,12-dioxo-, methyl ester (RTA-402; bardoxolone-methyl). (C, D) CDDO-Me increases expression of ARE-driven luciferase 18 hours after drug treatment in HBEC 3KT and HME1, respectively. Firefly ARE-luciferase normalized to renilla control (RLU). Mean ± SEM of 6 replicates, *p

Techniques Used: Activation Assay, Expressing, Luciferase

8) Product Images from "The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells"

Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2015/187265

Effects of XXT on Nrf2 and Keap1 mRNA expression levels in HUVECs.
Figure Legend Snippet: Effects of XXT on Nrf2 and Keap1 mRNA expression levels in HUVECs.

Techniques Used: Expressing

Effects of XXT on the protein expression levels of Keap1, Nrf2, HMOX1, GCLM, and NQO1 in HUVECs.
Figure Legend Snippet: Effects of XXT on the protein expression levels of Keap1, Nrf2, HMOX1, GCLM, and NQO1 in HUVECs.

Techniques Used: Expressing

Schematic representation of XXT activities on Keap1-Nrf2-ARE pathway.
Figure Legend Snippet: Schematic representation of XXT activities on Keap1-Nrf2-ARE pathway.

Techniques Used:

9) Product Images from "Hepatitis B virus stimulates G6PD expression through HBx-mediated Nrf2 activation"

Article Title: Hepatitis B virus stimulates G6PD expression through HBx-mediated Nrf2 activation

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.322

HBx-stimulated p62–Keap1 interaction is required for HBx-induced Nrf2 activation. ( a ) Localization of Dsred-Keap1 and Nrf2 in HBx-GFP-transfected Huh7 cells. ( b ) Colocalization of Dsred-Keap1 and p62 in HBx-GFP-expressing Huh7 cells. ( c ) Co-immunoprecipitation of Keap1 with p62 (left panel) or p62 with Keap1 (right panel) in HBx-expressing cells. ( d ) Immunostaining of p62 and Keap1 in normal liver tissues and HBV-infected non-tumor or tumor tissues. ( e ) Co-immunoprecipitation of HBx-GFP, p62, and Keap1 in HBx-GFP-expressing cells transfected without or with p62 siRNAs, Flag-p62, or Flag-p62T352A (p62TA). ( f ) Localization of Dsred-Keap1 in HBx-GFP-transfected cells with Flag-p62TA expression (upper panel) or p62 RNAi (lower panel). ( g ) Nqo1 mRNA level measured by qRT-PCR in HBx-GFP-expressing cells with or without p62 RNAi. ( h ) G6PD enzyme activity in cells treated as in ( g ). All the statistical data are presented as mean±S.E.M. of triplicate experiments. ** P
Figure Legend Snippet: HBx-stimulated p62–Keap1 interaction is required for HBx-induced Nrf2 activation. ( a ) Localization of Dsred-Keap1 and Nrf2 in HBx-GFP-transfected Huh7 cells. ( b ) Colocalization of Dsred-Keap1 and p62 in HBx-GFP-expressing Huh7 cells. ( c ) Co-immunoprecipitation of Keap1 with p62 (left panel) or p62 with Keap1 (right panel) in HBx-expressing cells. ( d ) Immunostaining of p62 and Keap1 in normal liver tissues and HBV-infected non-tumor or tumor tissues. ( e ) Co-immunoprecipitation of HBx-GFP, p62, and Keap1 in HBx-GFP-expressing cells transfected without or with p62 siRNAs, Flag-p62, or Flag-p62T352A (p62TA). ( f ) Localization of Dsred-Keap1 in HBx-GFP-transfected cells with Flag-p62TA expression (upper panel) or p62 RNAi (lower panel). ( g ) Nqo1 mRNA level measured by qRT-PCR in HBx-GFP-expressing cells with or without p62 RNAi. ( h ) G6PD enzyme activity in cells treated as in ( g ). All the statistical data are presented as mean±S.E.M. of triplicate experiments. ** P

Techniques Used: Activation Assay, Transfection, Expressing, Immunoprecipitation, Immunostaining, Infection, Quantitative RT-PCR, Activity Assay

HBx enhances G6PD activity via Nrf2. ( a and b ) Western blot ( a ) and qRT-PCR ( b ) analysis of G6PD expression in Huh7 cells expressing HBx-Myc. ( c ) The relative G6PD enzyme activity in cells treated as in ( a ). ( d – f ) The protein level ( d ), mRNA level ( e ), and enzyme activity ( f ) of G6PD in Huh7 cell with or without HBx-Myc expression and Nrf2 RNAi. All the statistical data are presented as mean±S.E.M. of triplicate experiments. * P
Figure Legend Snippet: HBx enhances G6PD activity via Nrf2. ( a and b ) Western blot ( a ) and qRT-PCR ( b ) analysis of G6PD expression in Huh7 cells expressing HBx-Myc. ( c ) The relative G6PD enzyme activity in cells treated as in ( a ). ( d – f ) The protein level ( d ), mRNA level ( e ), and enzyme activity ( f ) of G6PD in Huh7 cell with or without HBx-Myc expression and Nrf2 RNAi. All the statistical data are presented as mean±S.E.M. of triplicate experiments. * P

Techniques Used: Activity Assay, Western Blot, Quantitative RT-PCR, Expressing

HBV stimulates Nrf2 activation. ( a ) Immunostaining of Nrf2 in HepG2 and HepG2.2.15 cells. Note the nuclear distribution of Nrf2 in HepG2.2.15 cells. ( b ) The mRNA levels of Nqo1 , GSTm1 , and Cyp2a5 measured by qRT-PCR in HepG2 and HepG2.2.15 cells. ( c ) Huh7 cells transfected with HBV genomic DNA (HBV) or HBx-negative HBV genomic DNA (HBVX − ) were stained with HBcAg and Nrf2 antibodies at 48 h after transfection. ( d ) Huh7 cells were either treated with 250 μ M H 2 O 2 for 4 h or transfected with GFP or HBx-GFP for 48 h. Then, the cells were fixed and stained with Nrf2 antibody. ( e ) Western blot analysis of Nrf2 protein in nuclear extracts (N-Nrf2) and cell lysis (T-Nrf2) in cells treated as in ( d ). Lamin B was used as an internal control of nuclear fraction. ( f ) The mRNA levels of Nqo1 , GSTm1 , and Cyp2a5 measured by qRT-PCR in cells treated as in ( d ). ( g ) Nqo1 and GSTm1 mRNA levels in normal liver tissues and HBV-infected non-tumor or tumor tissues. All the statistical data are presented as mean±S.E.M. of triplicate experiments. ** P
Figure Legend Snippet: HBV stimulates Nrf2 activation. ( a ) Immunostaining of Nrf2 in HepG2 and HepG2.2.15 cells. Note the nuclear distribution of Nrf2 in HepG2.2.15 cells. ( b ) The mRNA levels of Nqo1 , GSTm1 , and Cyp2a5 measured by qRT-PCR in HepG2 and HepG2.2.15 cells. ( c ) Huh7 cells transfected with HBV genomic DNA (HBV) or HBx-negative HBV genomic DNA (HBVX − ) were stained with HBcAg and Nrf2 antibodies at 48 h after transfection. ( d ) Huh7 cells were either treated with 250 μ M H 2 O 2 for 4 h or transfected with GFP or HBx-GFP for 48 h. Then, the cells were fixed and stained with Nrf2 antibody. ( e ) Western blot analysis of Nrf2 protein in nuclear extracts (N-Nrf2) and cell lysis (T-Nrf2) in cells treated as in ( d ). Lamin B was used as an internal control of nuclear fraction. ( f ) The mRNA levels of Nqo1 , GSTm1 , and Cyp2a5 measured by qRT-PCR in cells treated as in ( d ). ( g ) Nqo1 and GSTm1 mRNA levels in normal liver tissues and HBV-infected non-tumor or tumor tissues. All the statistical data are presented as mean±S.E.M. of triplicate experiments. ** P

Techniques Used: Activation Assay, Immunostaining, Quantitative RT-PCR, Transfection, Staining, Western Blot, Lysis, Infection

Nrf2 and G6PD are required to HBx-promoted cell proliferation. ( a ) G6PD and Nrf2 protein levels in HBx-GFP cells with the expression of indicated shRNAs. ( b ) Cell proliferation assay of the cells in ( a ). Data are presented as mean±S.E.M. of triplicate experiments. ( c ) Colony forma tion assay of the cells in ( a ). Colonies in three randomly chosen fields per well were quantified. Data are presented as mean±S.E.M. of triplicate experiments. ** P
Figure Legend Snippet: Nrf2 and G6PD are required to HBx-promoted cell proliferation. ( a ) G6PD and Nrf2 protein levels in HBx-GFP cells with the expression of indicated shRNAs. ( b ) Cell proliferation assay of the cells in ( a ). Data are presented as mean±S.E.M. of triplicate experiments. ( c ) Colony forma tion assay of the cells in ( a ). Colonies in three randomly chosen fields per well were quantified. Data are presented as mean±S.E.M. of triplicate experiments. ** P

Techniques Used: Expressing, Proliferation Assay

10) Product Images from "Heme regulates gene expression by triggering Crm1-dependent nuclear export of Bach1"

Article Title: Heme regulates gene expression by triggering Crm1-dependent nuclear export of Bach1

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7600248

Regulation of DNA binding by heme in vivo . ( A ) GM02063 cells were transfected with the ho-1 reporter plasmid and indicated Bach1 and MafK expression plasmids. Cells were treated with or without hemin, and binding of Bach1 to the ho-1 E2 enhancer was examined by ChIP. ( B ) Binding of MafK or Nrf2 to the ho-1 E2 enhancer was examined as in (A). ( C ) Model describing the regulation of ho-1 or other target genes by Bach1 and heme. Besides MafK, other Maf-related factors may also serve as partners for Bach1. Bach1 occupies MARE enhancers to repress transcription under normal conditions. An increase in heme levels alleviates Bach1-mediated repression through inhibition of its DNA-binding activity and subsequent Crm1-dependent nuclear export, making MAREs available for activating Maf complexes including Nrf2 or p45 NF-E2.
Figure Legend Snippet: Regulation of DNA binding by heme in vivo . ( A ) GM02063 cells were transfected with the ho-1 reporter plasmid and indicated Bach1 and MafK expression plasmids. Cells were treated with or without hemin, and binding of Bach1 to the ho-1 E2 enhancer was examined by ChIP. ( B ) Binding of MafK or Nrf2 to the ho-1 E2 enhancer was examined as in (A). ( C ) Model describing the regulation of ho-1 or other target genes by Bach1 and heme. Besides MafK, other Maf-related factors may also serve as partners for Bach1. Bach1 occupies MARE enhancers to repress transcription under normal conditions. An increase in heme levels alleviates Bach1-mediated repression through inhibition of its DNA-binding activity and subsequent Crm1-dependent nuclear export, making MAREs available for activating Maf complexes including Nrf2 or p45 NF-E2.

Techniques Used: Binding Assay, In Vivo, Transfection, Plasmid Preparation, Expressing, Chromatin Immunoprecipitation, Inhibition, Activity Assay

11) Product Images from "Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: Role of NADPH oxidase"

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: Role of NADPH oxidase

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2014.03.015

NADPH oxidase-mediated CB1 upregulation requires Nrf2 activity in HSCs on SEA stimulation. (A) Immunofluorescence images showing the effect of SEA stimulation on the nuclear translocation of Nrf2 in normal HSCs with scram or Nox1/4 siRNAs ( n =6). (B) Nrf2 activity in scram, or Nox1/4 siRNAs-transfected HSCs with or without SEA stimulation ( n =6). (C) Western blot gels and summarized data showing the effects of Nrf2 siRNA on the protein expression of CB1 in normal HSCs with or without SEA stimulation ( n =4–6). * P
Figure Legend Snippet: NADPH oxidase-mediated CB1 upregulation requires Nrf2 activity in HSCs on SEA stimulation. (A) Immunofluorescence images showing the effect of SEA stimulation on the nuclear translocation of Nrf2 in normal HSCs with scram or Nox1/4 siRNAs ( n =6). (B) Nrf2 activity in scram, or Nox1/4 siRNAs-transfected HSCs with or without SEA stimulation ( n =6). (C) Western blot gels and summarized data showing the effects of Nrf2 siRNA on the protein expression of CB1 in normal HSCs with or without SEA stimulation ( n =4–6). * P

Techniques Used: Activity Assay, Immunofluorescence, Translocation Assay, Transfection, Western Blot, Expressing

12) Product Images from "An Autoregulatory Loop between Nrf2 and Cul3-Rbx1 Controls Their Cellular Abundance *"

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

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.121863

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

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

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

Techniques Used: Activity Assay

13) Product Images from "Nuclear Oncoprotein Prothymosin ? Is a Partner of Keap1: Implications for Expression of Oxidative Stress-Protecting Genes"

Article Title: Nuclear Oncoprotein Prothymosin ? Is a Partner of Keap1: Implications for Expression of Oxidative Stress-Protecting Genes

Journal:

doi: 10.1128/MCB.25.3.1089-1099.2005

ProTα competes with Nrf2 for binding to Keap1 in vitro. (A) Recombinant zz-Keap1 (50 ng) was immobilized on IgG-Sepharose and charged with 32 P-ProTα. Unbound ProTα was removed, and the immobilized Keap1-ProTα complex was
Figure Legend Snippet: ProTα competes with Nrf2 for binding to Keap1 in vitro. (A) Recombinant zz-Keap1 (50 ng) was immobilized on IgG-Sepharose and charged with 32 P-ProTα. Unbound ProTα was removed, and the immobilized Keap1-ProTα complex was

Techniques Used: Binding Assay, In Vitro, Recombinant

Impairment of the Keap1 NES function results in coordinated nuclear translocation of Keap1 and Nrf2 (A) and in transcriptional activation (B). (A) HeLa cells were cotransfected with the Nrf2- and either with the wild-type (wt) Keap1- or Keap1mNES-encoding
Figure Legend Snippet: Impairment of the Keap1 NES function results in coordinated nuclear translocation of Keap1 and Nrf2 (A) and in transcriptional activation (B). (A) HeLa cells were cotransfected with the Nrf2- and either with the wild-type (wt) Keap1- or Keap1mNES-encoding

Techniques Used: Translocation Assay, Activation Assay

14) Product Images from "Jun dimerization protein 2 is a critical component of the Nrf2/MafK complex regulating the response to ROS homeostasis"

Article Title: Jun dimerization protein 2 is a critical component of the Nrf2/MafK complex regulating the response to ROS homeostasis

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.448

Reciprocal interaction of JDP2, Nrf2 and MafK in vitro . ( a ) The structure of GST–JDP2 proteins used for EMSA assay in vitro . The structures of various mutant forms of JDP2 are shown below. ( b ) Expression of the GST–JDP2 proteins. The GST–JDP2 proteins were detected on 10% SDS-PAGE after purification with GST affinity resins. ( c ) EMSA assay of the JDP2 deletion mutants with human NQO1–ARE. EMSA assay was performed using a series of deletion mutants of the Jdp2 protein, as indicated in Materials and Methods. The upper bands of the DNA–protein complexes seem to be dimer forms. ( d ) The bZIP domain of JDP2 is recruited and binds MafK. The full-length rat MafK ( in vitro- translated MafK; IVT–MafK) was expressed using an in vitro transcription–translation system without [ 35 S]-methionine as recommended in the manufacturer's protocol. Affinity-resin-purified GST or GST–JDP2 fusion variant proteins were mixed with IVT–MafK. The bound MafK was applied to SDS-PAGE and immunodetected using a MafK-specific antibody. ( e ) The basic domain of JDP2 is recruited and binds Nrf2. The IVT–Nrf2 protein was incubated with GST–JDP2 and a protein-binding assay was performed as described in c . ( f ) The structure of mutant forms of various GST–MafK proteins used for EMSA assay in vitro . ( g ) Expression of the GST–MafK proteins. The GST–MafK was detected on 10% SDS-PAGE after purification with GST affinity resins. ( h ) The DNA-binding domain of MafK is recruited and binds JDP2. The full-length mouse JDP2 (IVT–JDP2) was expressed, using an in vitro transcription–translation system without [ 35 S]-methionine, as recommended in the manufacturer's protocol. Affinity-resin-purified GST or GST–MafK fusion variant proteins were mixed with IVT–JDP2. The bound JDP2 was applied to SDS-PAGE and immunodetected using a JDP2-specific antibody. ( i ) The ZIP domain of MafK is recruited and binds Nrf2. The structure and expression of GST–MafK and IVT–Nrf2 were described above. The bound Nrf2 was immunodetected using an Nrf2-specific antibody. ( j ) The structure of various mutant forms of the GST–Nrf2 protein used for EMSA assay in vitro . ( k ) Expression of the GST–Nrf2 protein. The GST–Nrf2 proteins were detected on 10% SDS-PAGE after purification with GST affinity resins. ( l ) The C-terminal and half-bZIP domain of Nrf2 are recruited and bind JDP2. The full-length mouse JDP2 (IVT-Jdp2) was expressed using an in vitro transcription–translation system without [ 35 S]-methionine. GST or GST–MafK fusion proteins were mixed with IVT–Jdp2. After washing, bound proteins were applied on SDS-PAGE and immunodetected using a Jdp2-specific antibody. ( m ) The bZIP and Neh 6 domains of Nrf2 are associated with MafK. The IVT–MafK protein and GST–Nrf2 proteins were incubated and the bound MafK was immunodetected using a MafK-specific antibody
Figure Legend Snippet: Reciprocal interaction of JDP2, Nrf2 and MafK in vitro . ( a ) The structure of GST–JDP2 proteins used for EMSA assay in vitro . The structures of various mutant forms of JDP2 are shown below. ( b ) Expression of the GST–JDP2 proteins. The GST–JDP2 proteins were detected on 10% SDS-PAGE after purification with GST affinity resins. ( c ) EMSA assay of the JDP2 deletion mutants with human NQO1–ARE. EMSA assay was performed using a series of deletion mutants of the Jdp2 protein, as indicated in Materials and Methods. The upper bands of the DNA–protein complexes seem to be dimer forms. ( d ) The bZIP domain of JDP2 is recruited and binds MafK. The full-length rat MafK ( in vitro- translated MafK; IVT–MafK) was expressed using an in vitro transcription–translation system without [ 35 S]-methionine as recommended in the manufacturer's protocol. Affinity-resin-purified GST or GST–JDP2 fusion variant proteins were mixed with IVT–MafK. The bound MafK was applied to SDS-PAGE and immunodetected using a MafK-specific antibody. ( e ) The basic domain of JDP2 is recruited and binds Nrf2. The IVT–Nrf2 protein was incubated with GST–JDP2 and a protein-binding assay was performed as described in c . ( f ) The structure of mutant forms of various GST–MafK proteins used for EMSA assay in vitro . ( g ) Expression of the GST–MafK proteins. The GST–MafK was detected on 10% SDS-PAGE after purification with GST affinity resins. ( h ) The DNA-binding domain of MafK is recruited and binds JDP2. The full-length mouse JDP2 (IVT–JDP2) was expressed, using an in vitro transcription–translation system without [ 35 S]-methionine, as recommended in the manufacturer's protocol. Affinity-resin-purified GST or GST–MafK fusion variant proteins were mixed with IVT–JDP2. The bound JDP2 was applied to SDS-PAGE and immunodetected using a JDP2-specific antibody. ( i ) The ZIP domain of MafK is recruited and binds Nrf2. The structure and expression of GST–MafK and IVT–Nrf2 were described above. The bound Nrf2 was immunodetected using an Nrf2-specific antibody. ( j ) The structure of various mutant forms of the GST–Nrf2 protein used for EMSA assay in vitro . ( k ) Expression of the GST–Nrf2 protein. The GST–Nrf2 proteins were detected on 10% SDS-PAGE after purification with GST affinity resins. ( l ) The C-terminal and half-bZIP domain of Nrf2 are recruited and bind JDP2. The full-length mouse JDP2 (IVT-Jdp2) was expressed using an in vitro transcription–translation system without [ 35 S]-methionine. GST or GST–MafK fusion proteins were mixed with IVT–Jdp2. After washing, bound proteins were applied on SDS-PAGE and immunodetected using a Jdp2-specific antibody. ( m ) The bZIP and Neh 6 domains of Nrf2 are associated with MafK. The IVT–MafK protein and GST–Nrf2 proteins were incubated and the bound MafK was immunodetected using a MafK-specific antibody

Techniques Used: In Vitro, Mutagenesis, Expressing, SDS Page, Purification, Variant Assay, Incubation, Protein Binding, Binding Assay

Cooperative binding of JDP2, MafK and Nrf2 to the ARE. ( a ) Tentative location of cis -elements in the ARE promoter region and its deletions mutants used for EMSA assay. AP-1-like (nt −471 to −466), ARE core (−462 to −456) and GC box (−455 to −453) elements are shown in white rectangular boxes. The deleted elements are shown in black rectangular boxes. The nucleotide sequences of the mutants (M1 to M7) were listed in the Supplementary Table I . ( b ) Competitive EMSA assay of JDP2 with ARE mutants. EMSA reactions were performed in the presence or absence of a competitive ARE as described in Materials and Methods, using [ γ - 32 P]-labeled double-stranded ARE oligonucleotides. GST–JDP2 and GST proteins were purified using GST affinity resins. ( c ) Competitive EMSA assay of MafK with ARE mutants. IVT–MafK proteins were incubated with an ARE probe in the presence or absence of competitive ARE mutants and EMSA assay was performed as described in Materials and Methods. IVT alone was used as a negative control. ( d ) Supershift EMSA assay with nuclear extracts (NEs) from WT MEFs and Jdp2 KO MEFs using the DNA probe of human NQO1–ARE. NEs from WT MEFs and Jdp2 KO MEFs were incubated without (lanes 1 and 6) and with antibodies specific for Jdp2 (lanes 2 and 7), Nrf2 (lanes 3 and 8), MafK (lanes 4 and 9) and IgG (lane 5 and 10). ‘Bound' indicates the supershifted DNA–protein complexes and ‘Free' indicates the ARE–DNA probe
Figure Legend Snippet: Cooperative binding of JDP2, MafK and Nrf2 to the ARE. ( a ) Tentative location of cis -elements in the ARE promoter region and its deletions mutants used for EMSA assay. AP-1-like (nt −471 to −466), ARE core (−462 to −456) and GC box (−455 to −453) elements are shown in white rectangular boxes. The deleted elements are shown in black rectangular boxes. The nucleotide sequences of the mutants (M1 to M7) were listed in the Supplementary Table I . ( b ) Competitive EMSA assay of JDP2 with ARE mutants. EMSA reactions were performed in the presence or absence of a competitive ARE as described in Materials and Methods, using [ γ - 32 P]-labeled double-stranded ARE oligonucleotides. GST–JDP2 and GST proteins were purified using GST affinity resins. ( c ) Competitive EMSA assay of MafK with ARE mutants. IVT–MafK proteins were incubated with an ARE probe in the presence or absence of competitive ARE mutants and EMSA assay was performed as described in Materials and Methods. IVT alone was used as a negative control. ( d ) Supershift EMSA assay with nuclear extracts (NEs) from WT MEFs and Jdp2 KO MEFs using the DNA probe of human NQO1–ARE. NEs from WT MEFs and Jdp2 KO MEFs were incubated without (lanes 1 and 6) and with antibodies specific for Jdp2 (lanes 2 and 7), Nrf2 (lanes 3 and 8), MafK (lanes 4 and 9) and IgG (lane 5 and 10). ‘Bound' indicates the supershifted DNA–protein complexes and ‘Free' indicates the ARE–DNA probe

Techniques Used: Binding Assay, Labeling, Purification, Incubation, Negative Control

JDP2 is required for ARE activation. ( a ) Protein detection in WT and Jdp2 KO MEFs using western blotting. The cellular lysates from WT and Jdp2 KO MEFs (40 μ g) were separated on SDS-PAGE and then transferred onto a membrane. Jdp2, MafK, Nrf2, HO-1, NQO1 and α -tubulin were immunodetected using specific antibodies. ( b ) Relative NQO1 promoter activity in WT and Jdp2 KO MEFs in the presence of TPA at the indicated exposure times. After 24 h of culture, TPA (10 –6 M) was added, transfectants with pGL4–hQR25–luciferase were incubated for an additional 48 h and luciferase activity was measured ( n =3) as described in Materials and Methods. ( c ) Effect of Nrf2 on NQO1 promoter activity. pGL4–hQR25–luciferase (400 ng) plus 0–100 ng of pcDNA3–Nrf2 were transfected into WT and Jdp2 KO MEFs (5 × 10 4 ). One day after transfection, cells were collected and luciferase activity was measured. Values from a representative experiment are given as mean±S.D. ( n =3). ( d ) Effect of JDP2 on ARE activity in the presence of Nrf2. Jdp2 KO MEFs (5 × 10 4 ) was transfected with 400 ng of pGL4–hQR25–luciferase, 50 ng of FLAG_S–Nrf2 and pcDNA–Jdp2, respectively, as indicated. One day after transfection, cells were collected and luciferase activity was measured ( n =3). ** P
Figure Legend Snippet: JDP2 is required for ARE activation. ( a ) Protein detection in WT and Jdp2 KO MEFs using western blotting. The cellular lysates from WT and Jdp2 KO MEFs (40 μ g) were separated on SDS-PAGE and then transferred onto a membrane. Jdp2, MafK, Nrf2, HO-1, NQO1 and α -tubulin were immunodetected using specific antibodies. ( b ) Relative NQO1 promoter activity in WT and Jdp2 KO MEFs in the presence of TPA at the indicated exposure times. After 24 h of culture, TPA (10 –6 M) was added, transfectants with pGL4–hQR25–luciferase were incubated for an additional 48 h and luciferase activity was measured ( n =3) as described in Materials and Methods. ( c ) Effect of Nrf2 on NQO1 promoter activity. pGL4–hQR25–luciferase (400 ng) plus 0–100 ng of pcDNA3–Nrf2 were transfected into WT and Jdp2 KO MEFs (5 × 10 4 ). One day after transfection, cells were collected and luciferase activity was measured. Values from a representative experiment are given as mean±S.D. ( n =3). ( d ) Effect of JDP2 on ARE activity in the presence of Nrf2. Jdp2 KO MEFs (5 × 10 4 ) was transfected with 400 ng of pGL4–hQR25–luciferase, 50 ng of FLAG_S–Nrf2 and pcDNA–Jdp2, respectively, as indicated. One day after transfection, cells were collected and luciferase activity was measured ( n =3). ** P

Techniques Used: Activation Assay, Western Blot, SDS Page, Activity Assay, Luciferase, Incubation, Transfection

JDP2 regulates the expression of Nrf2 target genes. ( a ) qPCR analysis of Nrf2 target genes in WT and Jdp2 KO MEFs. The expression levels determined are relative to the level of GAPDH. Error bars denote the S.D. from duplicate reactions by real-time PCR as described in Materials and Methods. The primer sequences of each Nrf2 target gene are listed in Supplementary Table SIII . * P
Figure Legend Snippet: JDP2 regulates the expression of Nrf2 target genes. ( a ) qPCR analysis of Nrf2 target genes in WT and Jdp2 KO MEFs. The expression levels determined are relative to the level of GAPDH. Error bars denote the S.D. from duplicate reactions by real-time PCR as described in Materials and Methods. The primer sequences of each Nrf2 target gene are listed in Supplementary Table SIII . * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

15) Product Images from "Nrf2 activation is associated with Z-DNA formation in the human HO-1 promoter"

Article Title: Nrf2 activation is associated with Z-DNA formation in the human HO-1 promoter

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt243

Human HO-1 gene promoter adopts a Z-DNA form in response to Nrf2-activating agent DEM. HeLa cells were transfected with the Z-probe (lanes 1 and 2) or Z mut -probe (lane 3) expression vectors. At 24 h after transfection, the cells were untreated (lane 1) or treated with 100 µM DEM for 3 h (lanes 2 and 3). ( A ) Whole-cell lysates were separated using SDS–PAGE, and the protein expression was analyzed through immunoblotting using specific antibodies, as indicated in the figure. ( B ) The gene expression was analyzed through real-time PCR using primers specific for the HO-1 and TXNRD1 genes. The value of lane 1 is set as 1, and the relative expression values are expressed as the means ± SEM of five independent assays. ( C ) A schematic figure of the human HO-1 and TXNRD1 gene loci and regions as detected through ChIP analysis. E2: E2 enhancer; E1: E1 enhancer; +1: transcription start site; ZZZ: HO-1 -TG repeat region; Pro: HO-1 gene promoter region; Ex3: HO-1 Exon 3 region; TXNRD1 -ARE: TXNRD1 ARE region. ( D ) HeLa cells were transfected with the Z-probe or Z mut -probe, and subsequently, the cells were exposed to 100 µM DEM for 3 h. After DEM treatment, a ChIP assay was performed using an anti-GFP antibody (black bars). Normal rabbit IgG was used as a negative control (gray bars). The amount of precipitated DNA was measured through real-time PCR using specific primer sets for the HO-1 -TG, HO-1 -Ex3 and TXNRD1 ARE regions. The anti-GFP ChIP value of lane 1 is set as 1, and the relative bindings are expressed as the means ± SEM of five independent assays. # P
Figure Legend Snippet: Human HO-1 gene promoter adopts a Z-DNA form in response to Nrf2-activating agent DEM. HeLa cells were transfected with the Z-probe (lanes 1 and 2) or Z mut -probe (lane 3) expression vectors. At 24 h after transfection, the cells were untreated (lane 1) or treated with 100 µM DEM for 3 h (lanes 2 and 3). ( A ) Whole-cell lysates were separated using SDS–PAGE, and the protein expression was analyzed through immunoblotting using specific antibodies, as indicated in the figure. ( B ) The gene expression was analyzed through real-time PCR using primers specific for the HO-1 and TXNRD1 genes. The value of lane 1 is set as 1, and the relative expression values are expressed as the means ± SEM of five independent assays. ( C ) A schematic figure of the human HO-1 and TXNRD1 gene loci and regions as detected through ChIP analysis. E2: E2 enhancer; E1: E1 enhancer; +1: transcription start site; ZZZ: HO-1 -TG repeat region; Pro: HO-1 gene promoter region; Ex3: HO-1 Exon 3 region; TXNRD1 -ARE: TXNRD1 ARE region. ( D ) HeLa cells were transfected with the Z-probe or Z mut -probe, and subsequently, the cells were exposed to 100 µM DEM for 3 h. After DEM treatment, a ChIP assay was performed using an anti-GFP antibody (black bars). Normal rabbit IgG was used as a negative control (gray bars). The amount of precipitated DNA was measured through real-time PCR using specific primer sets for the HO-1 -TG, HO-1 -Ex3 and TXNRD1 ARE regions. The anti-GFP ChIP value of lane 1 is set as 1, and the relative bindings are expressed as the means ± SEM of five independent assays. # P

Techniques Used: Transfection, Expressing, SDS Page, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control

Nrf2 activation is sufficient to induce Z-DNA formation in the human HO-1 -TG region. 293T-REx-FLAG-human Nrf2 cells stably expressing the Z-probe* were treated with Tet for the indicated periods of time. ( A ) Whole-cell lysates were separated using SDS–PAGE, and the protein expression was analyzed through immunoblotting using specific antibodies, as indicated in the figure. ( B ) The HO-1 gene expression after Tet administration was analyzed through real-time PCR using specific primer sets. The value at time 0 h was arbitrarily set as 1, and the relative expression values are expressed as the means ± SEM of three independent assays. ( C ) Nrf2 binding to the human HO-1 E2 enhancer ( HO-1 -E2) was analyzed through ChIP using an anti-FLAG antibody. ( D ) BRG1 recruitment to the human HO-1 gene promoter ( HO-1 -Pro) was analyzed through ChIP using an anti-BRG1 antibody. ( E ) Z-DNA formation was measured through real-time PCR using a specific primer set for the HO-1 TG repeat region ( HO-1 -TG). The value at 0 h is arbitrarily set as 1, and the relative bindings are expressed as the means ± SEM of three independent assays. # P
Figure Legend Snippet: Nrf2 activation is sufficient to induce Z-DNA formation in the human HO-1 -TG region. 293T-REx-FLAG-human Nrf2 cells stably expressing the Z-probe* were treated with Tet for the indicated periods of time. ( A ) Whole-cell lysates were separated using SDS–PAGE, and the protein expression was analyzed through immunoblotting using specific antibodies, as indicated in the figure. ( B ) The HO-1 gene expression after Tet administration was analyzed through real-time PCR using specific primer sets. The value at time 0 h was arbitrarily set as 1, and the relative expression values are expressed as the means ± SEM of three independent assays. ( C ) Nrf2 binding to the human HO-1 E2 enhancer ( HO-1 -E2) was analyzed through ChIP using an anti-FLAG antibody. ( D ) BRG1 recruitment to the human HO-1 gene promoter ( HO-1 -Pro) was analyzed through ChIP using an anti-BRG1 antibody. ( E ) Z-DNA formation was measured through real-time PCR using a specific primer set for the HO-1 TG repeat region ( HO-1 -TG). The value at 0 h is arbitrarily set as 1, and the relative bindings are expressed as the means ± SEM of three independent assays. # P

Techniques Used: Activation Assay, Stable Transfection, Expressing, SDS Page, Real-time Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation

Nrf2 knockdown reduces the DEM-induced Z-DNA formation in the human HO-1 -TG region. HeLa cells were transfected with the Z-probe expression vector, and at 24 h after transfection, the cells were transfected with control siRNA (Control) or Nrf2-specific siRNA (Nrf2) and treated with 100 µM DEM for 3 h. ( A ) Whole-cell lysates were separated using SDS–PAGE, and the protein expression was analyzed through immunoblotting using specific antibodies, as indicated in the figure. ( B ) HO-1 and TXNRD1 gene expression were analyzed through real-time PCR using specific primer sets. The value of lane 1 is arbitrarily set as 1, and the relative expressions are expressed as the means ± SEM of three independent assays. ( C and D ) A ChIP assay was performed using anti-GFP (C) or anti-BRG1 antibodies (black bars; D). Normal rabbit IgG was used as a negative control (gray bars). The Z-DNA formation was measured through real-time PCR using specific primer sets for the HO-1 -TG and TXNRD1 ARE regions (C). BRG1 binding was measured through real-time PCR using a specific primer set for the HO-1 gene promoter region ( HO-1 -Pro; D). The anti-GFP ChIP (C) or anti-BRG1 ChIP (D) values of lane 1 are set as 1, and the relative bindings are expressed as the means ± SEM of three independent assays. ** P
Figure Legend Snippet: Nrf2 knockdown reduces the DEM-induced Z-DNA formation in the human HO-1 -TG region. HeLa cells were transfected with the Z-probe expression vector, and at 24 h after transfection, the cells were transfected with control siRNA (Control) or Nrf2-specific siRNA (Nrf2) and treated with 100 µM DEM for 3 h. ( A ) Whole-cell lysates were separated using SDS–PAGE, and the protein expression was analyzed through immunoblotting using specific antibodies, as indicated in the figure. ( B ) HO-1 and TXNRD1 gene expression were analyzed through real-time PCR using specific primer sets. The value of lane 1 is arbitrarily set as 1, and the relative expressions are expressed as the means ± SEM of three independent assays. ( C and D ) A ChIP assay was performed using anti-GFP (C) or anti-BRG1 antibodies (black bars; D). Normal rabbit IgG was used as a negative control (gray bars). The Z-DNA formation was measured through real-time PCR using specific primer sets for the HO-1 -TG and TXNRD1 ARE regions (C). BRG1 binding was measured through real-time PCR using a specific primer set for the HO-1 gene promoter region ( HO-1 -Pro; D). The anti-GFP ChIP (C) or anti-BRG1 ChIP (D) values of lane 1 are set as 1, and the relative bindings are expressed as the means ± SEM of three independent assays. ** P

Techniques Used: Transfection, Expressing, Plasmid Preparation, SDS Page, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Binding Assay

Dynamics of Nrf2 and BRG1 on the HO-1 regulatory region in response to DEM. HeLa cells were transfected with the Z-probe expression vector, and at 24 h after transfection, the cells were treated with 100 µM DEM for various amounts of time, as indicated. ChIP assays were performed using anti-Nrf2 and anti-BRG1 antibodies. Nrf2 and BRG1 binding were measured through real-time PCR using specific primer sets for the HO-1 E2 enhancer ( HO-1 -E2; A ) or HO-1 gene promoter ( HO-1 -Pro; B ) regions. The value at 0 h is set as 1, and the relative changes are expressed as the means ± SEM of four independent assays. # P
Figure Legend Snippet: Dynamics of Nrf2 and BRG1 on the HO-1 regulatory region in response to DEM. HeLa cells were transfected with the Z-probe expression vector, and at 24 h after transfection, the cells were treated with 100 µM DEM for various amounts of time, as indicated. ChIP assays were performed using anti-Nrf2 and anti-BRG1 antibodies. Nrf2 and BRG1 binding were measured through real-time PCR using specific primer sets for the HO-1 E2 enhancer ( HO-1 -E2; A ) or HO-1 gene promoter ( HO-1 -Pro; B ) regions. The value at 0 h is set as 1, and the relative changes are expressed as the means ± SEM of four independent assays. # P

Techniques Used: Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction

16) Product Images from "Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin"

Article Title: Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2017.00017

Analysis of the binding of transcription regulators to the E2 and E1 sites of HMOX1 in wild-type and Arp5-KO cells . (A) In the absence of any oxidative stress (left panel), the repressor Bach1 forms a heterodimer with MafK, which binds to the Maf recognition element (MARE) at the E2 and E1 sites, and represses HMOX1 transcription. In the presence of oxidative stress (right panel), Bach1 is released, and the activator Nrf2 forms a heterodimer with MafK and binds to the MARE elements at E2 and E1. To analyze the binding of these transcriptional regulators, quantitative-ChIP assay was performed using antibodies against MafK (B) , Bach1 (C) , and Nrf2 (D) in wild-type (open bar) and Arp5-KO (filled bar) cells. The amount of immunoprecipitated fragment was normalized with respect to the input fraction value. Data shown are binding relative to that of exon 5. Averages from at least three independent experiments (± standard deviation) are shown. P -value (Student's t -test) for the difference between WT and Arp5 KO cells is indicated. * P
Figure Legend Snippet: Analysis of the binding of transcription regulators to the E2 and E1 sites of HMOX1 in wild-type and Arp5-KO cells . (A) In the absence of any oxidative stress (left panel), the repressor Bach1 forms a heterodimer with MafK, which binds to the Maf recognition element (MARE) at the E2 and E1 sites, and represses HMOX1 transcription. In the presence of oxidative stress (right panel), Bach1 is released, and the activator Nrf2 forms a heterodimer with MafK and binds to the MARE elements at E2 and E1. To analyze the binding of these transcriptional regulators, quantitative-ChIP assay was performed using antibodies against MafK (B) , Bach1 (C) , and Nrf2 (D) in wild-type (open bar) and Arp5-KO (filled bar) cells. The amount of immunoprecipitated fragment was normalized with respect to the input fraction value. Data shown are binding relative to that of exon 5. Averages from at least three independent experiments (± standard deviation) are shown. P -value (Student's t -test) for the difference between WT and Arp5 KO cells is indicated. * P

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Standard Deviation

17) Product Images from "Induction of human fetal hemoglobin via the NRF2 antioxidant response signaling pathway"

Article Title: Induction of human fetal hemoglobin via the NRF2 antioxidant response signaling pathway

Journal: Blood

doi: 10.1182/blood-2010-10-314096

tBHQ increases γ-globin mRNA and NRF2 binding to the γ-globin promoter in primary human erythroid cells. Human CD34 + peripheral blood cells were treated with tBHQ during in vitro erythroid differentiation. The effects of increasing concentrations
Figure Legend Snippet: tBHQ increases γ-globin mRNA and NRF2 binding to the γ-globin promoter in primary human erythroid cells. Human CD34 + peripheral blood cells were treated with tBHQ during in vitro erythroid differentiation. The effects of increasing concentrations

Techniques Used: Binding Assay, In Vitro

Hemin induces NRF2 to bind to the γ-globin promoter and requires NRF2 for full induction of γ-globin. (A) Effect of tBHQ and hemin on NQO1 and γ-globin mRNA levels. K562 cells were treated with 25μM of tBHQ and 20μM
Figure Legend Snippet: Hemin induces NRF2 to bind to the γ-globin promoter and requires NRF2 for full induction of γ-globin. (A) Effect of tBHQ and hemin on NQO1 and γ-globin mRNA levels. K562 cells were treated with 25μM of tBHQ and 20μM

Techniques Used:

tBHQ induces NRF2 nuclear translocation and binding of NRF2 to the γ-globin promoter in K562 cells. (A) Simplified schematic of the NRF2/ARE pathway. Under basal conditions, NRF2 is sequestered in the cytoplasm by KEAP1, which is an E3 ligase
Figure Legend Snippet: tBHQ induces NRF2 nuclear translocation and binding of NRF2 to the γ-globin promoter in K562 cells. (A) Simplified schematic of the NRF2/ARE pathway. Under basal conditions, NRF2 is sequestered in the cytoplasm by KEAP1, which is an E3 ligase

Techniques Used: Translocation Assay, Binding Assay

siRNA knockdown of NRF2 or mutation of the ARE element inhibit tBHQ induction of γ-globin gene expression. siRNA specific for NRF2 was transiently transfected into K562 cells. At 24 hours later, cells were treated with tBHQ for 12 hours before
Figure Legend Snippet: siRNA knockdown of NRF2 or mutation of the ARE element inhibit tBHQ induction of γ-globin gene expression. siRNA specific for NRF2 was transiently transfected into K562 cells. At 24 hours later, cells were treated with tBHQ for 12 hours before

Techniques Used: Mutagenesis, Expressing, Transfection

siRNA suppression of KEAP1 levels enhance NRF2 nuclear translocation and tBHQ induced γ-globin mRNA expression. siRNA specific for KEAP1 was transfected into K562 cells. At 24 hours later, cells were treated with tBHQ at 25μM for 24 hours
Figure Legend Snippet: siRNA suppression of KEAP1 levels enhance NRF2 nuclear translocation and tBHQ induced γ-globin mRNA expression. siRNA specific for KEAP1 was transfected into K562 cells. At 24 hours later, cells were treated with tBHQ at 25μM for 24 hours

Techniques Used: Translocation Assay, Expressing, Transfection

Induction of γ-globin gene expression by activators of the NRF2 antioxidant response element signaling pathway. (A) The human γ-globin gene promoter contains an ARE consensus sequence. (B) Compounds that activate NRF2/ARE pathway signaling.
Figure Legend Snippet: Induction of γ-globin gene expression by activators of the NRF2 antioxidant response element signaling pathway. (A) The human γ-globin gene promoter contains an ARE consensus sequence. (B) Compounds that activate NRF2/ARE pathway signaling.

Techniques Used: Expressing, Sequencing

18) Product Images from "Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network"

Article Title: Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0308083100

Competitive binding of Bach1 and Nrf2 regulates RNA polymerase recruitment and promoter clearance. ( A ) NIH 3T3 cells were treated with 10 μM hemin for 2 h, washed, and cultured further in hemin-free medium. Expression of HO-1 mRNA was determined
Figure Legend Snippet: Competitive binding of Bach1 and Nrf2 regulates RNA polymerase recruitment and promoter clearance. ( A ) NIH 3T3 cells were treated with 10 μM hemin for 2 h, washed, and cultured further in hemin-free medium. Expression of HO-1 mRNA was determined

Techniques Used: Binding Assay, Cell Culture, Expressing

19) Product Images from "Dual Biological Functions of a Cytoprotective Effect and Apoptosis Induction by Bioavailable Marine Carotenoid Fucoxanthinol through Modulation of the Nrf2 Activation in RAW264.7 Macrophage Cells"

Article Title: Dual Biological Functions of a Cytoprotective Effect and Apoptosis Induction by Bioavailable Marine Carotenoid Fucoxanthinol through Modulation of the Nrf2 Activation in RAW264.7 Macrophage Cells

Journal: Marine Drugs

doi: 10.3390/md15100305

Expression of HO-1 expression due to marine carotenoid, fucoxanthin (Fx) and its metabolite fucoxanthinol (FxOH) in RAW 264.7 macrophage cells. The HO-1 protein expression due to marine carotenoids (2.5–10 μM), the Fx and its metabolite FxOH effect on the Nrf2-ARE signaling in the cells was examined. ( a ) Western blot analysis of the HO-1 protein in the presence of compounds. ( b ) Densitometry analysis of the expression of HO-1 protein. Data were expressed as mean ( n = 3) ± SD. Analysis of data was carried out using ANOVA and Tukey’s method for multiple comparisons. A significance p
Figure Legend Snippet: Expression of HO-1 expression due to marine carotenoid, fucoxanthin (Fx) and its metabolite fucoxanthinol (FxOH) in RAW 264.7 macrophage cells. The HO-1 protein expression due to marine carotenoids (2.5–10 μM), the Fx and its metabolite FxOH effect on the Nrf2-ARE signaling in the cells was examined. ( a ) Western blot analysis of the HO-1 protein in the presence of compounds. ( b ) Densitometry analysis of the expression of HO-1 protein. Data were expressed as mean ( n = 3) ± SD. Analysis of data was carried out using ANOVA and Tukey’s method for multiple comparisons. A significance p

Techniques Used: Expressing, Western Blot

Suppression of the Bcl-xL protein expression due to marine carotenoids, fucoxanthin (Fx) and its metabolite fucoxanthinol (FxOH) in RAW 264.7 macrophage cells. The Bcl-xL protein expression with Fx and FxOH in the range of 2.5–20 μM on the Nrf2-ARE signaling in cells was examined. ( a ) Western blot analysis of the Bcl-xL protein in the presence of compounds. ( b ) Densitometry analysis of the expression of Bcl-xL protein. Data were expressed as mean ( n = 3) ± SD. Analysis of data was carried out using ANOVA and Tukey’s method for multiple comparisons. A significance p
Figure Legend Snippet: Suppression of the Bcl-xL protein expression due to marine carotenoids, fucoxanthin (Fx) and its metabolite fucoxanthinol (FxOH) in RAW 264.7 macrophage cells. The Bcl-xL protein expression with Fx and FxOH in the range of 2.5–20 μM on the Nrf2-ARE signaling in cells was examined. ( a ) Western blot analysis of the Bcl-xL protein in the presence of compounds. ( b ) Densitometry analysis of the expression of Bcl-xL protein. Data were expressed as mean ( n = 3) ± SD. Analysis of data was carried out using ANOVA and Tukey’s method for multiple comparisons. A significance p

Techniques Used: Expressing, Western Blot

Activation of Nrf2-ARE signaling due to marine carotenoids, fucoxanthin (Fx) and its metabolite fucoxanthinol (FxOH) in RAW 264.7 macrophage cells. The activation of Nrf2-ARE signaling due to marine carotenoids, the Fx (2.5–20 μM) and FxOH (2.5–10 μM) was evaluated by the reporter assay, as described in the text. Data were expressed as mean ( n = 4) ± SD. Analysis of data was carried out using ANOVA and Tukey’s method for multiple comparisons. Significance p
Figure Legend Snippet: Activation of Nrf2-ARE signaling due to marine carotenoids, fucoxanthin (Fx) and its metabolite fucoxanthinol (FxOH) in RAW 264.7 macrophage cells. The activation of Nrf2-ARE signaling due to marine carotenoids, the Fx (2.5–20 μM) and FxOH (2.5–10 μM) was evaluated by the reporter assay, as described in the text. Data were expressed as mean ( n = 4) ± SD. Analysis of data was carried out using ANOVA and Tukey’s method for multiple comparisons. Significance p

Techniques Used: Activation Assay, Reporter Assay

Expression of the nuclear Nrf2 protein due to marine carotenoids, fucoxanthin (Fx) and its metabolite fucoxanthinol (FxOH) in RAW 264.7 macrophage cells. The Nrf2 translocated into the nuclear in the presence of compounds (2.5 and 10 μM). ( a ) Western blot analysis of the Nrf2 protein in the presence of compounds. ( b ) Densitometry analysis of the expression of Nrf2 protein ( a ). Data were expressed as mean ( n = 3) ± SD. Analysis of data was carried out using ANOVA and Tukey’s method for multiple comparison. Significance p
Figure Legend Snippet: Expression of the nuclear Nrf2 protein due to marine carotenoids, fucoxanthin (Fx) and its metabolite fucoxanthinol (FxOH) in RAW 264.7 macrophage cells. The Nrf2 translocated into the nuclear in the presence of compounds (2.5 and 10 μM). ( a ) Western blot analysis of the Nrf2 protein in the presence of compounds. ( b ) Densitometry analysis of the expression of Nrf2 protein ( a ). Data were expressed as mean ( n = 3) ± SD. Analysis of data was carried out using ANOVA and Tukey’s method for multiple comparison. Significance p

Techniques Used: Expressing, Western Blot

Suppression of the phospho-Akt (pAkt) expression in the presence of marine carotenoids, fucoxanthin (Fx) and its metabolite fucoxanthinol (FxOH) in RAW 264.7 macrophage cells. The pAkt protein expression due to the Fx and FxOH in the range of 2.5–20 μM on the Nrf2-ARE signaling in cells was examined. ( a ) Western blot analysis of the pAkt protein in the presence of compounds. ( b ) Densitometry analysis of the expression of pAkt protein. Data were expressed as mean ( n = 3) ± SD. Analysis of data was carried out using ANOVA and Tukey’s method for multiple comparisons. A significance p
Figure Legend Snippet: Suppression of the phospho-Akt (pAkt) expression in the presence of marine carotenoids, fucoxanthin (Fx) and its metabolite fucoxanthinol (FxOH) in RAW 264.7 macrophage cells. The pAkt protein expression due to the Fx and FxOH in the range of 2.5–20 μM on the Nrf2-ARE signaling in cells was examined. ( a ) Western blot analysis of the pAkt protein in the presence of compounds. ( b ) Densitometry analysis of the expression of pAkt protein. Data were expressed as mean ( n = 3) ± SD. Analysis of data was carried out using ANOVA and Tukey’s method for multiple comparisons. A significance p

Techniques Used: Expressing, Western Blot

20) Product Images from "Nrf2-AKT interactions regulate heme oxygenase 1 expression in kidney epithelia during hypoxia and hypoxia-reoxygenation"

Article Title: Nrf2-AKT interactions regulate heme oxygenase 1 expression in kidney epithelia during hypoxia and hypoxia-reoxygenation

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00362.2016

Effects of chronic hypoxia on Nrf2-regulated antioxidant gene expression in kidney epithelia. HK-2 ( A ) and pMKE ( B ) cells were exposed to RA, 1% hypoxia for 12 h (0hR), or hypoxia-reoxygenation for 6 h (6hR), RNA was then isolated and subjected to qRT-PCR. Data are presented as means ± SE ( n = 3–4). *RA vs. hypoxia or hypoxia-reoxygenation. C : immunoblot analysis of HMOX1 expression in HK-2 ( left ) and pMKE ( right ) cells exposed to hypoxia and reoxygenation. The band intensity was quantitated using β-actin and shown from a representative blot ( n = 3). D : HIF1α and Nrf2 expression was analyzed by Western blot analysis in HK-2 and pMKE cells exposed to chronic hypoxia and hypoxia-reoxygenation. A representative blot of two independent experiments is shown.
Figure Legend Snippet: Effects of chronic hypoxia on Nrf2-regulated antioxidant gene expression in kidney epithelia. HK-2 ( A ) and pMKE ( B ) cells were exposed to RA, 1% hypoxia for 12 h (0hR), or hypoxia-reoxygenation for 6 h (6hR), RNA was then isolated and subjected to qRT-PCR. Data are presented as means ± SE ( n = 3–4). *RA vs. hypoxia or hypoxia-reoxygenation. C : immunoblot analysis of HMOX1 expression in HK-2 ( left ) and pMKE ( right ) cells exposed to hypoxia and reoxygenation. The band intensity was quantitated using β-actin and shown from a representative blot ( n = 3). D : HIF1α and Nrf2 expression was analyzed by Western blot analysis in HK-2 and pMKE cells exposed to chronic hypoxia and hypoxia-reoxygenation. A representative blot of two independent experiments is shown.

Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Western Blot

Effects of Nrf2 knockdown on hypoxia stimulated HMOX1 expression. qRT-PCR analysis of HMOX1 ( A ) and Nrf2 ( B ) mRNA expression in HK-2 cells transfected with either scrambled (Scr)-silent interfering (si) RNA or Nrf2-siRNA and subsequently exposed to acute or chronic hypoxia. Values from the Scr-siRNA transfected cells are considered as 1. *RA vs. hypoxia; **Scr-siRNA vs. Nrf2-SiRNA ( n = 3).
Figure Legend Snippet: Effects of Nrf2 knockdown on hypoxia stimulated HMOX1 expression. qRT-PCR analysis of HMOX1 ( A ) and Nrf2 ( B ) mRNA expression in HK-2 cells transfected with either scrambled (Scr)-silent interfering (si) RNA or Nrf2-siRNA and subsequently exposed to acute or chronic hypoxia. Values from the Scr-siRNA transfected cells are considered as 1. *RA vs. hypoxia; **Scr-siRNA vs. Nrf2-SiRNA ( n = 3).

Techniques Used: Expressing, Quantitative RT-PCR, Transfection

Hypoxia-inducible Hmox1 expression is blunted in Nrf2-deficient kidney epithelia. Primary kidney epithelial cells from Nrf2 +/+ or Nrf2 −/− mice were isolated and then exposed to RA, acute or chronic hypoxia, and hypoxia-reoxygenation conditions. Cells were lysed for RNA and protein extractions. A : Hmox1 expression analyzed by qRT-PCR analysis. *RA vs. hypoxia; ** Nrf2 +/+ vs. Nrf2 −/− . Data are presented as means ± SE ( n = 4). B : Hmox1 levels were analyzed by immunoblot analysis and quantified as detailed above. Values from a representative blot ( n = 2) are shown.
Figure Legend Snippet: Hypoxia-inducible Hmox1 expression is blunted in Nrf2-deficient kidney epithelia. Primary kidney epithelial cells from Nrf2 +/+ or Nrf2 −/− mice were isolated and then exposed to RA, acute or chronic hypoxia, and hypoxia-reoxygenation conditions. Cells were lysed for RNA and protein extractions. A : Hmox1 expression analyzed by qRT-PCR analysis. *RA vs. hypoxia; ** Nrf2 +/+ vs. Nrf2 −/− . Data are presented as means ± SE ( n = 4). B : Hmox1 levels were analyzed by immunoblot analysis and quantified as detailed above. Values from a representative blot ( n = 2) are shown.

Techniques Used: Expressing, Mouse Assay, Isolation, Quantitative RT-PCR

Effects of acute hypoxia on Nrf2-regulated antioxidant gene expression in kidney epithelia. HK-2 ( A ) and primary murine kidney epithelial (pMKE) ( B ) cells were exposed to room air (RA), 1% hypoxia for 2 h (0hR), or hypoxia-reoxygenation for 6 h (6hR). RNA was isolated and Nrf2 target gene expression was analyzed by quantitative RT-PCR (qRT-PCR). Data are presented as means ± SE ( n = 3–4). *RA vs. hypoxia or hypoxia-reoxygenation. C : immunoblot analysis of HMOX1 levels in HK-2 ( left ) and pMKE (right) cells exposed to hypoxia and hypoxia-reoxygenation. The band intensity was quantitated using β-actin as a reference, and the value of RA controls was considered as one unit. Relative fold change (RFC) shown is from a representative blot of three independent samples ( n = 3). D : immunoblot analysis of hypoxia-inducible factor 1α (HIF1α) and Nrf2 expression in HK-2 and pMKE cells exposed to hypoxia and hypoxia-reoxygenation. A representative blot of two independent experiments is shown.
Figure Legend Snippet: Effects of acute hypoxia on Nrf2-regulated antioxidant gene expression in kidney epithelia. HK-2 ( A ) and primary murine kidney epithelial (pMKE) ( B ) cells were exposed to room air (RA), 1% hypoxia for 2 h (0hR), or hypoxia-reoxygenation for 6 h (6hR). RNA was isolated and Nrf2 target gene expression was analyzed by quantitative RT-PCR (qRT-PCR). Data are presented as means ± SE ( n = 3–4). *RA vs. hypoxia or hypoxia-reoxygenation. C : immunoblot analysis of HMOX1 levels in HK-2 ( left ) and pMKE (right) cells exposed to hypoxia and hypoxia-reoxygenation. The band intensity was quantitated using β-actin as a reference, and the value of RA controls was considered as one unit. Relative fold change (RFC) shown is from a representative blot of three independent samples ( n = 3). D : immunoblot analysis of hypoxia-inducible factor 1α (HIF1α) and Nrf2 expression in HK-2 and pMKE cells exposed to hypoxia and hypoxia-reoxygenation. A representative blot of two independent experiments is shown.

Techniques Used: Expressing, Isolation, Quantitative RT-PCR

HMOX1 promoter activity and Nrf2 binding to the antioxidant response elements (AREs) in HK-2 cells exposed to hypoxia and hypoxia-reoxygenation. A : HMOX1 promoter reporter construct (100 ng) with pRL-TK plasmid (5 ng) were transfected into cells, exposed to hypoxia (0hR) and hypoxia-reoxygenation for 6 h (6hR). Luciferase activity was analyzed and expressed related to RA-exposed cells. *RA vs. exposure ( n = 3). B: Chromatin immunoprecipitation (ChIP) analysis of Nrf2 binding to the functional AREs of the HMOX1 promoter. HK-2 cells were exposed to RA, hypoxia, or hypoxia-reoxygenation, and chromatin was cross-linked and immunoprecipitated with IgG or anti-Nrf2 antibodies. Immunoprecipitated DNA was subjected to qRT-PCR using SYBR primers. Top : positions of the ARE sites and forward (F) and reverse (R) primers of the HMOX1 enhancer used in ChIP assays. Nrf2 binding to the HMOX1 promoter in cells exposed to acute ( left ) or chronic ( right ) hypoxia and hypoxia-reoxygenation. Data are presented as means ± SE ( n = 3). *RA vs. exposure.
Figure Legend Snippet: HMOX1 promoter activity and Nrf2 binding to the antioxidant response elements (AREs) in HK-2 cells exposed to hypoxia and hypoxia-reoxygenation. A : HMOX1 promoter reporter construct (100 ng) with pRL-TK plasmid (5 ng) were transfected into cells, exposed to hypoxia (0hR) and hypoxia-reoxygenation for 6 h (6hR). Luciferase activity was analyzed and expressed related to RA-exposed cells. *RA vs. exposure ( n = 3). B: Chromatin immunoprecipitation (ChIP) analysis of Nrf2 binding to the functional AREs of the HMOX1 promoter. HK-2 cells were exposed to RA, hypoxia, or hypoxia-reoxygenation, and chromatin was cross-linked and immunoprecipitated with IgG or anti-Nrf2 antibodies. Immunoprecipitated DNA was subjected to qRT-PCR using SYBR primers. Top : positions of the ARE sites and forward (F) and reverse (R) primers of the HMOX1 enhancer used in ChIP assays. Nrf2 binding to the HMOX1 promoter in cells exposed to acute ( left ) or chronic ( right ) hypoxia and hypoxia-reoxygenation. Data are presented as means ± SE ( n = 3). *RA vs. exposure.

Techniques Used: Activity Assay, Binding Assay, Construct, Plasmid Preparation, Transfection, Luciferase, Chromatin Immunoprecipitation, Functional Assay, Immunoprecipitation, Quantitative RT-PCR

21) Product Images from "Overexpression of HO-1 Protects against TNF-?-Mediated Airway Inflammation by Down-Regulation of TNFR1-Dependent Oxidative Stress"

Article Title: Overexpression of HO-1 Protects against TNF-?-Mediated Airway Inflammation by Down-Regulation of TNFR1-Dependent Oxidative Stress

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.090016

Overexpression of Nrf2-dependent HO-1 inhibits VCAM-1 and ICAM-1 expression in HTSMCs. Cells were pretreated with CoPP IX for 16 hours in the presence or absence of ZnPP IX, and then treated with TNF-α for ( A ) 24 hours or ( B ) 5 hours. The protein
Figure Legend Snippet: Overexpression of Nrf2-dependent HO-1 inhibits VCAM-1 and ICAM-1 expression in HTSMCs. Cells were pretreated with CoPP IX for 16 hours in the presence or absence of ZnPP IX, and then treated with TNF-α for ( A ) 24 hours or ( B ) 5 hours. The protein

Techniques Used: Over Expression, Expressing

Overexpression of Nrf2-dependent HO-1 inhibits NF-κB activation and translocation. A: Cells were stimulated with TNF-α for the indicated time intervals. The cell lysates were subjected to Western blot using an anti-phospho-p65 (Ser 536
Figure Legend Snippet: Overexpression of Nrf2-dependent HO-1 inhibits NF-κB activation and translocation. A: Cells were stimulated with TNF-α for the indicated time intervals. The cell lysates were subjected to Western blot using an anti-phospho-p65 (Ser 536

Techniques Used: Over Expression, Activation Assay, Translocation Assay, Western Blot

CoPP IX induces Nrf2-dependent HO-1 expression and activity in HTSMCs. A: Cells were incubated with CoPP IX for the indicated time intervals and HO-1 protein expression was determined by Western blot. B: Cells were incubated with 1 μmol/L CoPP
Figure Legend Snippet: CoPP IX induces Nrf2-dependent HO-1 expression and activity in HTSMCs. A: Cells were incubated with CoPP IX for the indicated time intervals and HO-1 protein expression was determined by Western blot. B: Cells were incubated with 1 μmol/L CoPP

Techniques Used: Expressing, Activity Assay, Incubation, Western Blot

A proposed pathway for overexpression of Nrf2-dependent HO-1 protects against TNF-α-induced oxidative stress and airway inflammation. TNF-α stimulates ROS production through TNFR1/c-Src/NADPH oxidase, in turn initiates the activation of
Figure Legend Snippet: A proposed pathway for overexpression of Nrf2-dependent HO-1 protects against TNF-α-induced oxidative stress and airway inflammation. TNF-α stimulates ROS production through TNFR1/c-Src/NADPH oxidase, in turn initiates the activation of

Techniques Used: Over Expression, Activation Assay

22) Product Images from "The Role of c-Jun Phosphorylation in EpRE Activation of Phase II Genes"

Article Title: The Role of c-Jun Phosphorylation in EpRE Activation of Phase II Genes

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2009.07.036

HNE increases the amount of endogenous nuclear Nrf2 in a time-dependent manner
Figure Legend Snippet: HNE increases the amount of endogenous nuclear Nrf2 in a time-dependent manner

Techniques Used:

Fig. 3A. Effect of HNE on the recruitment of transcription factors to EpRE of (A) nqo2, (B) gclc and (C) gclm in HepG2 cells. ChIP assays were performed to examined the interaction in vivo of Nrf2, c-Jun and p-c-Jun with EpRE that either treated or untreated
Figure Legend Snippet: Fig. 3A. Effect of HNE on the recruitment of transcription factors to EpRE of (A) nqo2, (B) gclc and (C) gclm in HepG2 cells. ChIP assays were performed to examined the interaction in vivo of Nrf2, c-Jun and p-c-Jun with EpRE that either treated or untreated

Techniques Used: Chromatin Immunoprecipitation, In Vivo

Interaction of Nrf2 with either c-Jun or p-c-Jun
Figure Legend Snippet: Interaction of Nrf2 with either c-Jun or p-c-Jun

Techniques Used:

Interaction of Nrf2 with either c-Jun or p-c-Jun
Figure Legend Snippet: Interaction of Nrf2 with either c-Jun or p-c-Jun

Techniques Used:

23) Product Images from "BRG1 Interacts with Nrf2 To Selectively Mediate HO-1 Induction in Response to Oxidative Stress ▿"

Article Title: BRG1 Interacts with Nrf2 To Selectively Mediate HO-1 Induction in Response to Oxidative Stress ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00700-06

In vivo binding of Nrf2 to AREs is independent of BRG1. SHCon or SH4 cells were treated with 100 μM DEM for 3.5 h and analyzed by ChIP with antibodies against Nrf2 and primers that amplify the HO-1 E1, HO-1 E2, HO-1 exon 3, NQO1 promoter (pr), or NQO1 exon 2 regions. Normal rabbit IgG was used as a control. The input lane represents PCR using 5% chromosomal DNA without immunoprecipitation.
Figure Legend Snippet: In vivo binding of Nrf2 to AREs is independent of BRG1. SHCon or SH4 cells were treated with 100 μM DEM for 3.5 h and analyzed by ChIP with antibodies against Nrf2 and primers that amplify the HO-1 E1, HO-1 E2, HO-1 exon 3, NQO1 promoter (pr), or NQO1 exon 2 regions. Normal rabbit IgG was used as a control. The input lane represents PCR using 5% chromosomal DNA without immunoprecipitation.

Techniques Used: In Vivo, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation

Nrf2 recruits BRG1 to both the HO-1 and NQO1 genes in response to DEM. (A) Schematic presentation of the primer locations used to amplify genomic regions. Human HO-1 E1 and E2 enhancer regions contain AREs, and the promoter region (pr) encompasses a TATA box. HO-1 exon 3 was used as a control. The human NQO1 promoter region (pr) harbors both an ARE and a TATA box. The genomic region encompassing NQO1 exon 2 was used as a control. (B) ChIP analyses of the HO-1 and NQO1 genes using antibodies against Nrf2 and BRG1. SHCon cells were treated with 100 μM DEM for 3.5 h, and ChIP analysis was performed with antibodies against Nrf2 and BRG1. Normal rabbit IgG was used as a control. (C) ChIP analyses of HO-1 and NQO1 genes in the presence of Nrf2 siRNA. SHCon cells were transfected with control siRNA (siCon) or Nrf2 siRNA (siNrf2) and treated with 100 μM DEM for 3.5 h. ChIP analysis was performed with antibodies against BRG1 and primers for HO-1 and NQO1 . In the input lane, 5% unprecipitated chromosomal DNA was amplified.
Figure Legend Snippet: Nrf2 recruits BRG1 to both the HO-1 and NQO1 genes in response to DEM. (A) Schematic presentation of the primer locations used to amplify genomic regions. Human HO-1 E1 and E2 enhancer regions contain AREs, and the promoter region (pr) encompasses a TATA box. HO-1 exon 3 was used as a control. The human NQO1 promoter region (pr) harbors both an ARE and a TATA box. The genomic region encompassing NQO1 exon 2 was used as a control. (B) ChIP analyses of the HO-1 and NQO1 genes using antibodies against Nrf2 and BRG1. SHCon cells were treated with 100 μM DEM for 3.5 h, and ChIP analysis was performed with antibodies against Nrf2 and BRG1. Normal rabbit IgG was used as a control. (C) ChIP analyses of HO-1 and NQO1 genes in the presence of Nrf2 siRNA. SHCon cells were transfected with control siRNA (siCon) or Nrf2 siRNA (siNrf2) and treated with 100 μM DEM for 3.5 h. ChIP analysis was performed with antibodies against BRG1 and primers for HO-1 and NQO1 . In the input lane, 5% unprecipitated chromosomal DNA was amplified.

Techniques Used: Chromatin Immunoprecipitation, Transfection, Amplification

Knockdown of Nrf2 decreases DEM-inducible expression of HO-1 and NQO1 . (A) Immunoblot analysis of BRG1 and hBRM in SW480 and SW13 cells. Nuclear extracts of SW480 and SW13 cells were analyzed by anti-BRG1, anti-hBRM, or anti-lamin B antibodies. (B) SW480 cells were transfected with control siRNA (siCon) or Nrf2 siRNA (siNrf2). At 24 h posttransfection, cells were treated with 100 μM DEM for 4 h and then subjected to immunoblot analysis using anti-Nrf2 (upper panel) or anti-lamin B (lower panel) antibodies. (C) HO-1 and NQO1 expression in siCon and siNrf2 cells after DEM treatment. SW480 cells were transfected with siCon or siNrf2. Following treatment with 100 μM DEM for the indicated time periods, total RNA was isolated and HO-1 and NQO1 mRNA expression was determined by quantitative RT-PCR, with 18S rRNA used as an internal standard. The means of three independent experiments performed in duplicate are shown, and error bars represent SE. HO-1 and NQO1 expression in siCon cells without DEM treatment was set at 1.
Figure Legend Snippet: Knockdown of Nrf2 decreases DEM-inducible expression of HO-1 and NQO1 . (A) Immunoblot analysis of BRG1 and hBRM in SW480 and SW13 cells. Nuclear extracts of SW480 and SW13 cells were analyzed by anti-BRG1, anti-hBRM, or anti-lamin B antibodies. (B) SW480 cells were transfected with control siRNA (siCon) or Nrf2 siRNA (siNrf2). At 24 h posttransfection, cells were treated with 100 μM DEM for 4 h and then subjected to immunoblot analysis using anti-Nrf2 (upper panel) or anti-lamin B (lower panel) antibodies. (C) HO-1 and NQO1 expression in siCon and siNrf2 cells after DEM treatment. SW480 cells were transfected with siCon or siNrf2. Following treatment with 100 μM DEM for the indicated time periods, total RNA was isolated and HO-1 and NQO1 mRNA expression was determined by quantitative RT-PCR, with 18S rRNA used as an internal standard. The means of three independent experiments performed in duplicate are shown, and error bars represent SE. HO-1 and NQO1 expression in siCon cells without DEM treatment was set at 1.

Techniques Used: Expressing, Transfection, Isolation, Quantitative RT-PCR

Schematic presentation of the role that BRG1 and Nrf2 play in the DEM-inducible expression of HO-1 . In response to oxidative stress, Nrf2 accumulates in the nucleus. Subsequently, Nrf2 recruits BRG1 to the HO-1 regulatory region and facilitates chromatin remodeling through interactions with BRG1. BRG1 remodels the nucleosome (shown in red) to generate Z-DNA formation. Formation of Z-DNA facilitates the opening of chromatin and the recruitment of RNA Pol II.
Figure Legend Snippet: Schematic presentation of the role that BRG1 and Nrf2 play in the DEM-inducible expression of HO-1 . In response to oxidative stress, Nrf2 accumulates in the nucleus. Subsequently, Nrf2 recruits BRG1 to the HO-1 regulatory region and facilitates chromatin remodeling through interactions with BRG1. BRG1 remodels the nucleosome (shown in red) to generate Z-DNA formation. Formation of Z-DNA facilitates the opening of chromatin and the recruitment of RNA Pol II.

Techniques Used: Expressing

) and Flag-BRG1 expression plasmids. Whole-cell lysates were immunoprecipitated with anti-Flag antibody-conjugated beads, followed by immunoblot analysis with anti-Nrf2 and anti-Flag antibodies (lanes 5 to 8). Nrf2 and BRG1 expression levels were verified by immunoblot analysis with anti-Nrf2 and anti-Flag antibodies (lanes 1 to 4). (D) Schematic presentation of the GBD-Nrf2 fusion proteins. (E to G) BRG1, but not the BRG1 K785A mutant (BRG1m), enhances Nrf2 transactivation activity in SW13 cells. SW13 cells were transfected with 20 ng of pCEP4 Gal4-Luc and 100 ng of GBD-NT (E), GBD-Neh2-4 (F), or GBD-Neh5 (G), along with 400 ng or 800 ng of the BRG1 or 800 ng of the BRG1 K785 mutant (BRG1m) expression plasmids. Luciferase activity of the reporter vector alone was set at 1, and relative values from three independent experiments each carried out in duplicate are shown with SE. *, significantly different from the activity of Gal4-Nrf2 alone ( P
Figure Legend Snippet: ) and Flag-BRG1 expression plasmids. Whole-cell lysates were immunoprecipitated with anti-Flag antibody-conjugated beads, followed by immunoblot analysis with anti-Nrf2 and anti-Flag antibodies (lanes 5 to 8). Nrf2 and BRG1 expression levels were verified by immunoblot analysis with anti-Nrf2 and anti-Flag antibodies (lanes 1 to 4). (D) Schematic presentation of the GBD-Nrf2 fusion proteins. (E to G) BRG1, but not the BRG1 K785A mutant (BRG1m), enhances Nrf2 transactivation activity in SW13 cells. SW13 cells were transfected with 20 ng of pCEP4 Gal4-Luc and 100 ng of GBD-NT (E), GBD-Neh2-4 (F), or GBD-Neh5 (G), along with 400 ng or 800 ng of the BRG1 or 800 ng of the BRG1 K785 mutant (BRG1m) expression plasmids. Luciferase activity of the reporter vector alone was set at 1, and relative values from three independent experiments each carried out in duplicate are shown with SE. *, significantly different from the activity of Gal4-Nrf2 alone ( P

Techniques Used: Expressing, Immunoprecipitation, Mutagenesis, Activity Assay, Transfection, Luciferase, Plasmid Preparation

BRG1 knockdown selectively downregulates DEM-inducible expression of HO-1 . (A) Immunoblot analysis of BRG1 knockdown cell lines. SHCon, SH4, and SH7 cells were treated with 100 μM DEM for 4 h, and the nuclear extracts were subjected to immunoblot analysis with antibodies against BRG1, hBRM, Nrf2, or lamin B. (B and C) RNA blot analysis of BRG1 knockdown cell lines. SHCon, SH4, and SH7 cells were treated with 100 μM DEM for the indicated time periods, and total RNA was isolated and analyzed by RNA blotting for HO-1 , NQO1 , and GAPDH (B). Band intensity was quantified by NIH Image and plotted after normalization with the GAPDH signal (C). The maximal induction level in SHCon cells was set at 100, and the mean relative expression levels from two independent experiments at each time point are presented. (D) Quantitative RT-PCR analysis of GCSL , GCSH , and AKR1C1 . The induction levels of the GCSL , GCSH , and AKR1C1 genes at the 12-h time point in SHCon cells were set at 100, and the means of relative expression from two independent experiments each carried out in duplicate at each time point are presented; 18S rRNA was used as an internal standard.
Figure Legend Snippet: BRG1 knockdown selectively downregulates DEM-inducible expression of HO-1 . (A) Immunoblot analysis of BRG1 knockdown cell lines. SHCon, SH4, and SH7 cells were treated with 100 μM DEM for 4 h, and the nuclear extracts were subjected to immunoblot analysis with antibodies against BRG1, hBRM, Nrf2, or lamin B. (B and C) RNA blot analysis of BRG1 knockdown cell lines. SHCon, SH4, and SH7 cells were treated with 100 μM DEM for the indicated time periods, and total RNA was isolated and analyzed by RNA blotting for HO-1 , NQO1 , and GAPDH (B). Band intensity was quantified by NIH Image and plotted after normalization with the GAPDH signal (C). The maximal induction level in SHCon cells was set at 100, and the mean relative expression levels from two independent experiments at each time point are presented. (D) Quantitative RT-PCR analysis of GCSL , GCSH , and AKR1C1 . The induction levels of the GCSL , GCSH , and AKR1C1 genes at the 12-h time point in SHCon cells were set at 100, and the means of relative expression from two independent experiments each carried out in duplicate at each time point are presented; 18S rRNA was used as an internal standard.

Techniques Used: Expressing, Northern blot, Isolation, Quantitative RT-PCR

TG repeats can be replaced with an alternative Z-DNA-forming sequence to sustain induction of the hHO-1 gene. (A) The sequence of the human HO-1 promoter with TG repeats. The numbers above and below the sequence are relative positions from the transcription start site (+1). (B) Schematic representation of the mutant reporter construct. Thirty TG repeats are replaced with 18 GC repeats or the NQO1 fifth exon (random). (C) Activities of the pCEP4 hHO-1 luc (WT), 18GC, and random constructs. The pCEP4 hHO-1 luc, 18GC, and random constructs were cotransfected with the Nrf2 expression plasmid into SW480 cells. The means of three independent experiments each carried out in duplicate are shown, and error bars represent SE. Luciferase activity in the WT HO-1 promoter reporter plasmid alone was set at 1. *, significantly different from the activity of the WT construct in the presence of the same amount of Nrf2 expression plasmid ( P
Figure Legend Snippet: TG repeats can be replaced with an alternative Z-DNA-forming sequence to sustain induction of the hHO-1 gene. (A) The sequence of the human HO-1 promoter with TG repeats. The numbers above and below the sequence are relative positions from the transcription start site (+1). (B) Schematic representation of the mutant reporter construct. Thirty TG repeats are replaced with 18 GC repeats or the NQO1 fifth exon (random). (C) Activities of the pCEP4 hHO-1 luc (WT), 18GC, and random constructs. The pCEP4 hHO-1 luc, 18GC, and random constructs were cotransfected with the Nrf2 expression plasmid into SW480 cells. The means of three independent experiments each carried out in duplicate are shown, and error bars represent SE. Luciferase activity in the WT HO-1 promoter reporter plasmid alone was set at 1. *, significantly different from the activity of the WT construct in the presence of the same amount of Nrf2 expression plasmid ( P

Techniques Used: Sequencing, Mutagenesis, Construct, Expressing, Plasmid Preparation, Luciferase, Activity Assay

24) Product Images from "Development of Novel Nrf2/ARE Inducers Bearing Pyrazino[2,1-a]isoquinolin Scaffold with Potent In Vitro Efficacy and Enhanced Physicochemical Properties"

Article Title: Development of Novel Nrf2/ARE Inducers Bearing Pyrazino[2,1-a]isoquinolin Scaffold with Potent In Vitro Efficacy and Enhanced Physicochemical Properties

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

doi: 10.3390/molecules22091541

3g can elevate the protein level of Nrf2 and NQO1.
Figure Legend Snippet: 3g can elevate the protein level of Nrf2 and NQO1.

Techniques Used:

25) Product Images from "A SIRT1 agonist reduces cognitive decline in type 2 diabetic rats through antioxidative and anti-inflammatory mechanisms"

Article Title: A SIRT1 agonist reduces cognitive decline in type 2 diabetic rats through antioxidative and anti-inflammatory mechanisms

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2018.9699

Effect of a Nrf2 agonist on the antioxidative action of SRT1720 in PC12 diabetic model cells. Densitometric analysis was performed on western blotting results to quantify (A) HO-1 and (B) Nrf2 protein expression in control, SRT1720-treated and SRT1720 + Nrf2 agonist-treated PC12 diabetic model cells. (C) Representative western blot bands for HO-1 and Nrf2 protein expression in each group. Curcumin was employed as the Nrf2 agonist. **P
Figure Legend Snippet: Effect of a Nrf2 agonist on the antioxidative action of SRT1720 in PC12 diabetic model cells. Densitometric analysis was performed on western blotting results to quantify (A) HO-1 and (B) Nrf2 protein expression in control, SRT1720-treated and SRT1720 + Nrf2 agonist-treated PC12 diabetic model cells. (C) Representative western blot bands for HO-1 and Nrf2 protein expression in each group. Curcumin was employed as the Nrf2 agonist. **P

Techniques Used: Western Blot, Expressing

Effect of SRT1720 on DM-induced alterations in HSP70, SIRT1, Nrf2 and HO-1 protein expression in hippocampal tissue. (A) Protein expression levels of HSP70, SIRT1, Nrf2 and HO-1 were detected by western blot analysis. Densitometric analysis of western blotting results was performed to quantify the protein levels of (B) HSP70, (C) SIRT1, (D) Nrf2 and (E) HO-1 in control, DM and SRT1720-treated DM rats. The results demonstrated that alterations observed in DM rats compared with control rats were reversed by SRT1720 treatment. **P
Figure Legend Snippet: Effect of SRT1720 on DM-induced alterations in HSP70, SIRT1, Nrf2 and HO-1 protein expression in hippocampal tissue. (A) Protein expression levels of HSP70, SIRT1, Nrf2 and HO-1 were detected by western blot analysis. Densitometric analysis of western blotting results was performed to quantify the protein levels of (B) HSP70, (C) SIRT1, (D) Nrf2 and (E) HO-1 in control, DM and SRT1720-treated DM rats. The results demonstrated that alterations observed in DM rats compared with control rats were reversed by SRT1720 treatment. **P

Techniques Used: Expressing, Western Blot

Effect of a Nrf2 agonist on GSH-PX, GSH, SOD and MDA levels in PC12 diabetic model cells treated with SRT1720. ELISA was performed to determine the levels of (A) GSH-PX, (B) GSH, (C) SOD and (D) MDA in control, SRT1720-treated and SRT1720 + Nrf2 agonist-treated PC12 diabetic model cells. Curcumin was employed as the Nrf2 agonist. **P
Figure Legend Snippet: Effect of a Nrf2 agonist on GSH-PX, GSH, SOD and MDA levels in PC12 diabetic model cells treated with SRT1720. ELISA was performed to determine the levels of (A) GSH-PX, (B) GSH, (C) SOD and (D) MDA in control, SRT1720-treated and SRT1720 + Nrf2 agonist-treated PC12 diabetic model cells. Curcumin was employed as the Nrf2 agonist. **P

Techniques Used: Multiple Displacement Amplification, Enzyme-linked Immunosorbent Assay

26) Product Images from "Distal Airways in Mice Exposed to Cigarette Smoke"

Article Title: Distal Airways in Mice Exposed to Cigarette Smoke

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2007-0295OC

CS-induced antioxidant and detoxification gene expression is primarily regulated by the Nrf2 transcription factor. ( A ) C22 cells were transfected with siRNA duplexes targeting the expression of the Nrf2 or NF-κB transcription factors, or a scrambled siRNA duplex. Two days after transfection, RNAs were isolated and subjected to real-time RT-PCR for expression of Nrf2 or NF-κB, respectively. Data are representative of at least three independent experiments ± SEM. * P
Figure Legend Snippet: CS-induced antioxidant and detoxification gene expression is primarily regulated by the Nrf2 transcription factor. ( A ) C22 cells were transfected with siRNA duplexes targeting the expression of the Nrf2 or NF-κB transcription factors, or a scrambled siRNA duplex. Two days after transfection, RNAs were isolated and subjected to real-time RT-PCR for expression of Nrf2 or NF-κB, respectively. Data are representative of at least three independent experiments ± SEM. * P

Techniques Used: Expressing, Transfection, Isolation, Quantitative RT-PCR

Chronic CS exposure increases Nrf2 expression in terminal bronchiolar epithelium. ( A ) RNAs isolated from laser capture microdissection–retrieved cells from terminal bronchiolar region of mice exposed to CS for various times were subjected to real-time RT-PCR using oligonucleotides specific for murine Nrf2. Data are representative of three separate experiments using two independently isolated RNA samples per time point. Expression of Nrf2 was normalized to that of the β2-MG housekeeping gene ± SEM. * P
Figure Legend Snippet: Chronic CS exposure increases Nrf2 expression in terminal bronchiolar epithelium. ( A ) RNAs isolated from laser capture microdissection–retrieved cells from terminal bronchiolar region of mice exposed to CS for various times were subjected to real-time RT-PCR using oligonucleotides specific for murine Nrf2. Data are representative of three separate experiments using two independently isolated RNA samples per time point. Expression of Nrf2 was normalized to that of the β2-MG housekeeping gene ± SEM. * P

Techniques Used: Expressing, Isolation, Laser Capture Microdissection, Mouse Assay, Quantitative RT-PCR

Exposure of C22 cells to CS extract results in Nrf2 translocation to the nucleus and stabilization, but does not increase Nrf2 expression. ( A ) RNA isolated from C22 cells exposed to serum-free media alone or containing 10% CS extract for up to 24 hours were subjected to real-time RT-PCR using Nrf2-specific oligonucleotides. After normalization to β2-MG, results are expressed as fold change above untreated conditions. Data are representative of three independent experiments ± SEM. ( B ) Western blot analysis of whole cell protein extracts collected from C22 cells exposed to CS for up to 24 hours using an Nrf2-specific antibody. The same membrane was also probed with a GAPDH-specific antibody to control for loading. Data are representative of at least three independent experiments. ( C ) C22 cells were exposed to serum-free media containing 10% CS extract for up to 1 hour. Cells were fixed and stained using a Nrf2-specific antibody. ( D ) C22 cells were exposed to serum-free media containing 10% CS extract for up to 1 hour and the amount of Nrf2 located in the cytoplasmic (C) and nuclear (N) fractions was determined by Western blot analysis using an Nrf2-specific antibody. Data are representative of at least three independent experiments.
Figure Legend Snippet: Exposure of C22 cells to CS extract results in Nrf2 translocation to the nucleus and stabilization, but does not increase Nrf2 expression. ( A ) RNA isolated from C22 cells exposed to serum-free media alone or containing 10% CS extract for up to 24 hours were subjected to real-time RT-PCR using Nrf2-specific oligonucleotides. After normalization to β2-MG, results are expressed as fold change above untreated conditions. Data are representative of three independent experiments ± SEM. ( B ) Western blot analysis of whole cell protein extracts collected from C22 cells exposed to CS for up to 24 hours using an Nrf2-specific antibody. The same membrane was also probed with a GAPDH-specific antibody to control for loading. Data are representative of at least three independent experiments. ( C ) C22 cells were exposed to serum-free media containing 10% CS extract for up to 1 hour. Cells were fixed and stained using a Nrf2-specific antibody. ( D ) C22 cells were exposed to serum-free media containing 10% CS extract for up to 1 hour and the amount of Nrf2 located in the cytoplasmic (C) and nuclear (N) fractions was determined by Western blot analysis using an Nrf2-specific antibody. Data are representative of at least three independent experiments.

Techniques Used: Translocation Assay, Expressing, Isolation, Quantitative RT-PCR, Western Blot, Staining

27) Product Images from "Acerogenin A from Acer nikoense Maxim Prevents Oxidative Stress-Induced Neuronal Cell Death through Nrf2-Mediated Heme Oxygenase-1 Expression in Mouse Hippocampal HT22 Cell Line"

Article Title: Acerogenin A from Acer nikoense Maxim Prevents Oxidative Stress-Induced Neuronal Cell Death through Nrf2-Mediated Heme Oxygenase-1 Expression in Mouse Hippocampal HT22 Cell Line

Journal: Molecules

doi: 10.3390/molecules200712545

The effects of acerogenin A on the nuclear translocation of Nrf2 ( A ) and Nrf2-mediated HO-1 ( B ) in HT22 cells. HT22 cells were treated with 30 μM of acerogenin A for 0, 30, 60, and 90 min ( A ). The nuclei were fractionated from the cytosol by using PER-Mammalian Protein Extraction Buffer, as described in the Experimental Section ( A ). HT22 cells were transiently transfected with Nrf2 siRNA and then treated with 30 μM acerogenin A for 12 h ( B ). Western blot analysis was performed, and representative blots of three independent experiments are shown. Data are presented as the mean value ± SD values of three independent experiments. * p
Figure Legend Snippet: The effects of acerogenin A on the nuclear translocation of Nrf2 ( A ) and Nrf2-mediated HO-1 ( B ) in HT22 cells. HT22 cells were treated with 30 μM of acerogenin A for 0, 30, 60, and 90 min ( A ). The nuclei were fractionated from the cytosol by using PER-Mammalian Protein Extraction Buffer, as described in the Experimental Section ( A ). HT22 cells were transiently transfected with Nrf2 siRNA and then treated with 30 μM acerogenin A for 12 h ( B ). Western blot analysis was performed, and representative blots of three independent experiments are shown. Data are presented as the mean value ± SD values of three independent experiments. * p

Techniques Used: Translocation Assay, Protein Extraction, Transfection, Western Blot

28) Product Images from "Marine Peroxy Sesquiterpenoids Induce Apoptosis by Modulation of Nrf2-ARE Signaling in HCT116 Colon Cancer Cells"

Article Title: Marine Peroxy Sesquiterpenoids Induce Apoptosis by Modulation of Nrf2-ARE Signaling in HCT116 Colon Cancer Cells

Journal: Marine Drugs

doi: 10.3390/md16100347

Expression of the nuclear factor-erythroid-2-related factor (Nrf2) protein due to peroxy sesquiterpenoids in HCT116 colon cancer cells. ( a ) Western blot analysis of the Nrf2 protein in the presence of compounds 1 and 2 (above) and densitometry analysis of the expression of the Nrf2 protein (below). Data were expressed as means ± SD. A Student’s t -test was applied to analyze the significance of the difference. * p
Figure Legend Snippet: Expression of the nuclear factor-erythroid-2-related factor (Nrf2) protein due to peroxy sesquiterpenoids in HCT116 colon cancer cells. ( a ) Western blot analysis of the Nrf2 protein in the presence of compounds 1 and 2 (above) and densitometry analysis of the expression of the Nrf2 protein (below). Data were expressed as means ± SD. A Student’s t -test was applied to analyze the significance of the difference. * p

Techniques Used: Expressing, Western Blot

29) Product Images from "c-Myc is a Nrf2-interacting protein that negatively regulates phase II genes through their electrophile responsive elements"

Article Title: c-Myc is a Nrf2-interacting protein that negatively regulates phase II genes through their electrophile responsive elements

Journal: IUBMB life

doi: 10.1002/iub.314

Recruitment of c-Myc to EpRE promoter. c-Myc interacts with the promoter of A. GCLM (EpRE) B. NQO1 (EpRE) and C. GCLC (EpRE4) under both basal and induced conditions in HBE1 cells. ChIP assays were performed to examine the in vivo interaction of c-Myc with EpRE that either treated or untreated with 10 μM HNE. In panel B and C cells were transfected with 50 nM c-Myc-siRNA for 24 h or treated with HNE for 3 h. Vehicle and non-specific siRNA was used in the controls. After stimulation with HNE or transfection with si-RNA, cells were fixed with 1% formaldehyde, lysed, and sonicated to shear chromatin in 0.2-0.8-kb fragments, which were then immunoprecipitated with anti c-Myc antibody or anti Nrf2 antibody. Purified DNA was then analyzed with RT-PCR with specific primers amplifying EpRE elements. Results are indicating a representative experiment out of four.
Figure Legend Snippet: Recruitment of c-Myc to EpRE promoter. c-Myc interacts with the promoter of A. GCLM (EpRE) B. NQO1 (EpRE) and C. GCLC (EpRE4) under both basal and induced conditions in HBE1 cells. ChIP assays were performed to examine the in vivo interaction of c-Myc with EpRE that either treated or untreated with 10 μM HNE. In panel B and C cells were transfected with 50 nM c-Myc-siRNA for 24 h or treated with HNE for 3 h. Vehicle and non-specific siRNA was used in the controls. After stimulation with HNE or transfection with si-RNA, cells were fixed with 1% formaldehyde, lysed, and sonicated to shear chromatin in 0.2-0.8-kb fragments, which were then immunoprecipitated with anti c-Myc antibody or anti Nrf2 antibody. Purified DNA was then analyzed with RT-PCR with specific primers amplifying EpRE elements. Results are indicating a representative experiment out of four.

Techniques Used: Chromatin Immunoprecipitation, In Vivo, Transfection, Sonication, Immunoprecipitation, Purification, Reverse Transcription Polymerase Chain Reaction

Interaction of c-Myc with either Nrf2 or p-c-Jun at both basal and induced conditions. HBE1 cells were treated with 10 μM HNE or vehicle control for 3 hours. Nuclear proteins were immunoprecipitated with the indicated antibodies: A. c-Myc. B. Nrf2. C. p-c-Jun, and visualized by Western blot analysis with anti Nrf2, anti c-Myc, anti p-c-Jun, anti p-c-Myc or anti c-Jun antibodies. Lamin was used as loading control and identified with anti-lamin antibody. Expression levels of the indicated proteins in the whole cell extract are shown in total lysate (lower panels).
Figure Legend Snippet: Interaction of c-Myc with either Nrf2 or p-c-Jun at both basal and induced conditions. HBE1 cells were treated with 10 μM HNE or vehicle control for 3 hours. Nuclear proteins were immunoprecipitated with the indicated antibodies: A. c-Myc. B. Nrf2. C. p-c-Jun, and visualized by Western blot analysis with anti Nrf2, anti c-Myc, anti p-c-Jun, anti p-c-Myc or anti c-Jun antibodies. Lamin was used as loading control and identified with anti-lamin antibody. Expression levels of the indicated proteins in the whole cell extract are shown in total lysate (lower panels).

Techniques Used: Immunoprecipitation, Western Blot, Expressing

Knockdown of c-Myc up- regulates transcription of self defense genes. A. c-Myc knockdown decreased the protein level of c-Myc. Cells were transfected with 50 nM c-Myc-siRNA or control siRNA for 48 h. c-Myc and Nrf2 proteins were then detected by western blot analyses. B. c-Myc knockdown increases the expression of GCLC (black columns) and GCLM (net columns) mRNA. HBE1 cells were transfected with control si-RNA or 50 nM c-Myc-siRNA. Following 24 h of transfection cells were treated or untreated with HNE for 18 h and mRNA expression was measured. Cells were also treated with 10 μM HNE alone. C. c-Myc knockdown increases the expression of NQO1 (black columns) and NQO2 (net columns) mRNA. HBE1 cells were transfected with control siRNA or 50 nM c-Myc–siRNA. Following 24 h transfection cells were treated or untreated with HNE for 18 h. Cells were treated with 10 μM HNE alone and mRNA expression were analyzed by RT-PCR. C. Results are reported as mean ± S.E for four independent experiments. * p
Figure Legend Snippet: Knockdown of c-Myc up- regulates transcription of self defense genes. A. c-Myc knockdown decreased the protein level of c-Myc. Cells were transfected with 50 nM c-Myc-siRNA or control siRNA for 48 h. c-Myc and Nrf2 proteins were then detected by western blot analyses. B. c-Myc knockdown increases the expression of GCLC (black columns) and GCLM (net columns) mRNA. HBE1 cells were transfected with control si-RNA or 50 nM c-Myc-siRNA. Following 24 h of transfection cells were treated or untreated with HNE for 18 h and mRNA expression was measured. Cells were also treated with 10 μM HNE alone. C. c-Myc knockdown increases the expression of NQO1 (black columns) and NQO2 (net columns) mRNA. HBE1 cells were transfected with control siRNA or 50 nM c-Myc–siRNA. Following 24 h transfection cells were treated or untreated with HNE for 18 h. Cells were treated with 10 μM HNE alone and mRNA expression were analyzed by RT-PCR. C. Results are reported as mean ± S.E for four independent experiments. * p

Techniques Used: Transfection, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

c-Myc knock down stabilized Nrf2 by increasing its half-life. HBE1 control cells A. or transfected with 50 nM c-Myc-siRNA B. were treated with 25 μM cyclohexamide (CHX) at 48 h post transfection and incubated for the time periods indicated in figure. Total proteins were collected and endogenous Nrf2 was detected by immunoblot analysis, and the intensity of the Nrf2 bands was quantified compared to actin. The amount of Nrf2 before addition of CHX was set as 1. Blot shown is a representative experiment. Graph shows the results as mean ± S.E. of 3 determination. * p
Figure Legend Snippet: c-Myc knock down stabilized Nrf2 by increasing its half-life. HBE1 control cells A. or transfected with 50 nM c-Myc-siRNA B. were treated with 25 μM cyclohexamide (CHX) at 48 h post transfection and incubated for the time periods indicated in figure. Total proteins were collected and endogenous Nrf2 was detected by immunoblot analysis, and the intensity of the Nrf2 bands was quantified compared to actin. The amount of Nrf2 before addition of CHX was set as 1. Blot shown is a representative experiment. Graph shows the results as mean ± S.E. of 3 determination. * p

Techniques Used: Transfection, Incubation

30) Product Images from "The Heme Oxygenase-1 Protein Is Overexpressed in Human Renal Cancer Cells following Activation of the Ras-Raf-ERK Pathway and Mediates Anti-Apoptotic Signal *"

Article Title: The Heme Oxygenase-1 Protein Is Overexpressed in Human Renal Cancer Cells following Activation of the Ras-Raf-ERK Pathway and Mediates Anti-Apoptotic Signal *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.248401

Nrf2 is overexpressed in RCC, and plays an important role in HO-1 expression. A , representative photomicrographs show the expression of Nrf2 in human RCC and normal kidney tissues detected by immunohistochemistry. Brown color dots , expression of Nrf2.
Figure Legend Snippet: Nrf2 is overexpressed in RCC, and plays an important role in HO-1 expression. A , representative photomicrographs show the expression of Nrf2 in human RCC and normal kidney tissues detected by immunohistochemistry. Brown color dots , expression of Nrf2.

Techniques Used: Expressing, Immunohistochemistry

Nrf2 is important for H-Ras- and ERK-induced HO-1 transcription: A , 786-0 cells were transfected (overnight) with either different concentrations of H-Ras(12V) or empty vector ( left panel ); treated with either PD98059 (50 μ m ) or vehicle alone
Figure Legend Snippet: Nrf2 is important for H-Ras- and ERK-induced HO-1 transcription: A , 786-0 cells were transfected (overnight) with either different concentrations of H-Ras(12V) or empty vector ( left panel ); treated with either PD98059 (50 μ m ) or vehicle alone

Techniques Used: Transfection, Plasmid Preparation

31) Product Images from "Sestrin2 is induced by glucose starvation via the unfolded protein response and protects cells from non-canonical necroptotic cell death"

Article Title: Sestrin2 is induced by glucose starvation via the unfolded protein response and protects cells from non-canonical necroptotic cell death

Journal: Scientific Reports

doi: 10.1038/srep22538

Glucose starvation activates SESN2 via NRF2- and ATF4- dependent but via p53-independent mechanism. ( a,b ) Silencing of either NRF2 or ATF4 inhibits SESN2 activation by glucose withdrawal. H1299 cells were infected with lentiviral vectors expressing shNRF2, shATF4 or control shLuciferase (shLuc). The cells were incubated with glucose-free medium for 12 hr and expression of the corresponding proteins was examined by immunoblotting ( a ) and qPCR ( b ). The data represent a mean of three independent experiments ± S.D. Data in ( b ) were analyzed with two-way ANOVA followed by linear contrasts with Bonferroni correction (adjusted α = 0.05/9 = 0.0056, **P
Figure Legend Snippet: Glucose starvation activates SESN2 via NRF2- and ATF4- dependent but via p53-independent mechanism. ( a,b ) Silencing of either NRF2 or ATF4 inhibits SESN2 activation by glucose withdrawal. H1299 cells were infected with lentiviral vectors expressing shNRF2, shATF4 or control shLuciferase (shLuc). The cells were incubated with glucose-free medium for 12 hr and expression of the corresponding proteins was examined by immunoblotting ( a ) and qPCR ( b ). The data represent a mean of three independent experiments ± S.D. Data in ( b ) were analyzed with two-way ANOVA followed by linear contrasts with Bonferroni correction (adjusted α = 0.05/9 = 0.0056, **P

Techniques Used: Activation Assay, Infection, Expressing, Incubation, Real-time Polymerase Chain Reaction

32) Product Images from "Activation of NRF2 by Nitrosative Agents and H2O2 Involves KEAP1 Disulfide Formation *"

Article Title: Activation of NRF2 by Nitrosative Agents and H2O2 Involves KEAP1 Disulfide Formation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.051714

Oxidation of KEAP1 parallels NRF2 stabilization. HeLa cells transfected with pcDNA-Myc-His-KEAP1 or Cys-mutants derivatives (1 μg), pCI-HA-NRF2 (1.5 μg), and peYFP-N1 (0.3 μg), as indicated, were treated with H 2 O 2 ( A , 0.2 m m ) for
Figure Legend Snippet: Oxidation of KEAP1 parallels NRF2 stabilization. HeLa cells transfected with pcDNA-Myc-His-KEAP1 or Cys-mutants derivatives (1 μg), pCI-HA-NRF2 (1.5 μg), and peYFP-N1 (0.3 μg), as indicated, were treated with H 2 O 2 ( A , 0.2 m m ) for

Techniques Used: Transfection

33) Product Images from "Quercitrin from Toona sinensis (Juss.) M.Roem. Attenuates Acetaminophen-Induced Acute Liver Toxicity in HepG2 Cells and Mice through Induction of Antioxidant Machinery and Inhibition of Inflammation"

Article Title: Quercitrin from Toona sinensis (Juss.) M.Roem. Attenuates Acetaminophen-Induced Acute Liver Toxicity in HepG2 Cells and Mice through Induction of Antioxidant Machinery and Inhibition of Inflammation

Journal: Nutrients

doi: 10.3390/nu8070431

Effects of quercitrin on expressions of Nrf2 and NQO1 in APAP intoxicated mice. The protein levels of Nrf2 ( A ) and NQO1 ( B ) were measured by Western blot analysis as described in Materials and Methods section. Protein expression levels were normalized with β-actin. All data represent means ± SD of five mice in each group. Significant differences were ( # ) p
Figure Legend Snippet: Effects of quercitrin on expressions of Nrf2 and NQO1 in APAP intoxicated mice. The protein levels of Nrf2 ( A ) and NQO1 ( B ) were measured by Western blot analysis as described in Materials and Methods section. Protein expression levels were normalized with β-actin. All data represent means ± SD of five mice in each group. Significant differences were ( # ) p

Techniques Used: Mouse Assay, Western Blot, Expressing

Up-regulation of nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE)-mediated phase II detoxifying enzyme in APAP-treated HepG2 cells. ( A ) Effect of quercitrin on protein expression of Nrf2; ( B ) Effect of quercitrin on ARE-luciferase activity; ( C ) Effect of quercitrin on protein level of quinone oxidoreductase 1 (NQO1). Luciferase activity was normalized with total protein content and expressed as fold induction of normal control. Protein expression levels were normalized with β-actin. All data represent means ± SD of at least three independent experiments. Significant differences were ( ## ) p
Figure Legend Snippet: Up-regulation of nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE)-mediated phase II detoxifying enzyme in APAP-treated HepG2 cells. ( A ) Effect of quercitrin on protein expression of Nrf2; ( B ) Effect of quercitrin on ARE-luciferase activity; ( C ) Effect of quercitrin on protein level of quinone oxidoreductase 1 (NQO1). Luciferase activity was normalized with total protein content and expressed as fold induction of normal control. Protein expression levels were normalized with β-actin. All data represent means ± SD of at least three independent experiments. Significant differences were ( ## ) p

Techniques Used: Expressing, Luciferase, Activity Assay

34) Product Images from "Inhibitory effects of hydrogen sulphide on pulmonary fibrosis in smoking rats via attenuation of oxidative stress and inflammation"

Article Title: Inhibitory effects of hydrogen sulphide on pulmonary fibrosis in smoking rats via attenuation of oxidative stress and inflammation

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12254

Representative immunoblots and densitometric analysis of Nrf2 in nucleus and cytosol (A) and its downstream targets HO-1 and Trx-1 (B). * P
Figure Legend Snippet: Representative immunoblots and densitometric analysis of Nrf2 in nucleus and cytosol (A) and its downstream targets HO-1 and Trx-1 (B). * P

Techniques Used: Western Blot

35) Product Images from "Glutathione transferases P1/P2 regulate the timing of signaling pathway activations and cell cycle progression during mouse liver regeneration"

Article Title: Glutathione transferases P1/P2 regulate the timing of signaling pathway activations and cell cycle progression during mouse liver regeneration

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.562

Differential mRNA expression of antioxidant enzymes in the livers of Gstp1/2 +/+ and Gstp1/2 −/− mice after PH. ( a ) mRNA of Nrf2 , MnSOD , catalase , Hmox-1 , Hsp70 , Hif1α , eNOS and iNOS expression in Gstp1/2 +/+ and Gstp1/2 −/− mice at the indicated times after PH. Results are expressed as fold induction compared with the Gstp1/2 +/+ control liver arbitrarily set at 1 and as mean±S.E.M. ( n =3–7 mice/group/time point). * P ≤0.05, ** P ≤0.01, Gstp1/2 −/− versus Gstp1/2 +/+ mice. ( b ) Pool of total proteins from three mice per time point were used for western blotting analysis of eNOS and iNOS in Gstp1/2 +/+ and Gstp1/2 −/− mice at the indicated times after PH. HSC70 is used as a loading control
Figure Legend Snippet: Differential mRNA expression of antioxidant enzymes in the livers of Gstp1/2 +/+ and Gstp1/2 −/− mice after PH. ( a ) mRNA of Nrf2 , MnSOD , catalase , Hmox-1 , Hsp70 , Hif1α , eNOS and iNOS expression in Gstp1/2 +/+ and Gstp1/2 −/− mice at the indicated times after PH. Results are expressed as fold induction compared with the Gstp1/2 +/+ control liver arbitrarily set at 1 and as mean±S.E.M. ( n =3–7 mice/group/time point). * P ≤0.05, ** P ≤0.01, Gstp1/2 −/− versus Gstp1/2 +/+ mice. ( b ) Pool of total proteins from three mice per time point were used for western blotting analysis of eNOS and iNOS in Gstp1/2 +/+ and Gstp1/2 −/− mice at the indicated times after PH. HSC70 is used as a loading control

Techniques Used: Expressing, Mouse Assay, Western Blot

Hepatic localization of NRF2 and MnSOD after PH in Gstp1/2 +/+ and Gstp1/2 −/− mice. Immunolocalization of NRF2 and MnSOD in the Gstp1/2 +/+ and Gstp1/2 −/− livers prior to PH (0 h) and at 24, 36 and 48 h after PH. Bars: 200 μ m
Figure Legend Snippet: Hepatic localization of NRF2 and MnSOD after PH in Gstp1/2 +/+ and Gstp1/2 −/− mice. Immunolocalization of NRF2 and MnSOD in the Gstp1/2 +/+ and Gstp1/2 −/− livers prior to PH (0 h) and at 24, 36 and 48 h after PH. Bars: 200 μ m

Techniques Used: Mouse Assay

36) Product Images from "KMS99220 Exerts Anti-Inflammatory Effects, Activates the Nrf2 Signaling and Interferes with IKK, JNK and p38 MAPK via HO-1"

Article Title: KMS99220 Exerts Anti-Inflammatory Effects, Activates the Nrf2 Signaling and Interferes with IKK, JNK and p38 MAPK via HO-1

Journal: Molecules and Cells

doi: 10.14348/molcells.2019.0129

KMS99220 activates the Nrf2 signaling pathway and induces HO-1 production in microglia (A–I) BV-2 cells were treated with various concentrations of KMS99220. (A) Nuclear extracts from BV-2 cells were obtained after incubation with KMS99220 for 3 h, and the amount of nuclear Nrf2 (57 kDa) was analyzed by Western blot analysis, using lamin B (67 kDa) as an internal control. After incubation with KMS99220 for 24 h, Western blot analysis was performed for HO-1 (32 kDa) (B), NQO1 (31 kDa) (C), GCLC (73 kDa) (D), and GCLM (31 kDa) (E) using β-actin as an internal control. After incubation for 6 h, RT-PCR was performed for HO-1 (F), NQO1 (G), GCLC (H), and GCLM (I) using GAPDH as an internal control. The data are expressed as fold changes compared with untreated control ± SEM (n = 3); # P
Figure Legend Snippet: KMS99220 activates the Nrf2 signaling pathway and induces HO-1 production in microglia (A–I) BV-2 cells were treated with various concentrations of KMS99220. (A) Nuclear extracts from BV-2 cells were obtained after incubation with KMS99220 for 3 h, and the amount of nuclear Nrf2 (57 kDa) was analyzed by Western blot analysis, using lamin B (67 kDa) as an internal control. After incubation with KMS99220 for 24 h, Western blot analysis was performed for HO-1 (32 kDa) (B), NQO1 (31 kDa) (C), GCLC (73 kDa) (D), and GCLM (31 kDa) (E) using β-actin as an internal control. After incubation for 6 h, RT-PCR was performed for HO-1 (F), NQO1 (G), GCLC (H), and GCLM (I) using GAPDH as an internal control. The data are expressed as fold changes compared with untreated control ± SEM (n = 3); # P

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

37) Product Images from "Could the Combination of Two Non-Psychotropic Cannabinoids Counteract Neuroinflammation? Effectiveness of Cannabidiol Associated with Cannabigerol"

Article Title: Could the Combination of Two Non-Psychotropic Cannabinoids Counteract Neuroinflammation? Effectiveness of Cannabidiol Associated with Cannabigerol

Journal: Medicina

doi: 10.3390/medicina55110747

Western blot analysis for Nrf2 shows an increase of Nrf2 nuclear translocation in the groups treated with CBD alone at 2.5 and 5 µM doses and in combination with CBG at a 5 µM dose. **** p
Figure Legend Snippet: Western blot analysis for Nrf2 shows an increase of Nrf2 nuclear translocation in the groups treated with CBD alone at 2.5 and 5 µM doses and in combination with CBG at a 5 µM dose. **** p

Techniques Used: Western Blot, Translocation Assay

38) Product Images from "Coordinate Regulation of Glutathione Biosynthesis and Release by Nrf2-Expressing Glia Potently Protects Neurons from Oxidative Stress"

Article Title: Coordinate Regulation of Glutathione Biosynthesis and Release by Nrf2-Expressing Glia Potently Protects Neurons from Oxidative Stress

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.23-08-03394.2003

Cortical glia have higher basal Nrf2 expression and ARE promotor activity than neurons. A , Western blot of heterologously expressed Nrf2 (105 kDa) and Nrf2DN (28 kDa) in HEK293 cells. B, A comigrating 105 kDa band corresponding to Nrf2 is observed in enriched cortical glial, but not neuronal, cultures. Densitometric analysis reveals an ∼12-fold difference in Nrf2 protein ( n = 3); * p
Figure Legend Snippet: Cortical glia have higher basal Nrf2 expression and ARE promotor activity than neurons. A , Western blot of heterologously expressed Nrf2 (105 kDa) and Nrf2DN (28 kDa) in HEK293 cells. B, A comigrating 105 kDa band corresponding to Nrf2 is observed in enriched cortical glial, but not neuronal, cultures. Densitometric analysis reveals an ∼12-fold difference in Nrf2 protein ( n = 3); * p

Techniques Used: Expressing, Activity Assay, Western Blot

39) Product Images from "Lack of aprataxin impairs mitochondrial functions via downregulation of the APE1/NRF1/NRF2 pathway"

Article Title: Lack of aprataxin impairs mitochondrial functions via downregulation of the APE1/NRF1/NRF2 pathway

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddv183

APE1, NRF1 and NRF2 in AOA1 fibroblasts and APTX-depleted Hela cells. APE1 ( A and D ), NRF1 ( B and E ) and NRF2 ( C and F ) protein levels are reduced in APTX-mutant (A–C) and APTX-depleted cells (D–F). Representative western blot. Values
Figure Legend Snippet: APE1, NRF1 and NRF2 in AOA1 fibroblasts and APTX-depleted Hela cells. APE1 ( A and D ), NRF1 ( B and E ) and NRF2 ( C and F ) protein levels are reduced in APTX-mutant (A–C) and APTX-depleted cells (D–F). Representative western blot. Values

Techniques Used: Mutagenesis, Western Blot

NRF1, NRF2, PDSS1 and SDHA levels in APE1-depleted cells. NRF1, NRF2, PDSS1 and SDHA mRNA ( A ) and protein levels ( B ) are decreased in APE1-interfered clones. Values are expressed as mean ± SEM in relative percentage of controls (* P
Figure Legend Snippet: NRF1, NRF2, PDSS1 and SDHA levels in APE1-depleted cells. NRF1, NRF2, PDSS1 and SDHA mRNA ( A ) and protein levels ( B ) are decreased in APE1-interfered clones. Values are expressed as mean ± SEM in relative percentage of controls (* P

Techniques Used: Clone Assay

Effects of AICAR ( A ) and rosiglitazone ( B ) supplementation on NRF1, NRF2, PDSS1, SDHA and TOM20 levels (B), in AOA1 fibroblasts. Values are expressed as mean ± SEM in relative percentage of controls (* P
Figure Legend Snippet: Effects of AICAR ( A ) and rosiglitazone ( B ) supplementation on NRF1, NRF2, PDSS1, SDHA and TOM20 levels (B), in AOA1 fibroblasts. Values are expressed as mean ± SEM in relative percentage of controls (* P

Techniques Used:

40) Product Images from "Protective Effects of L-Malate against Myocardial Ischemia/Reperfusion Injury in Rats"

Article Title: Protective Effects of L-Malate against Myocardial Ischemia/Reperfusion Injury in Rats

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2016/3803657

L-malate (LM) increases expression of total Nrf2 and nuclear Nrf2 protein and HO-1, NQO-1 protein after MIRI and decreases expression of Keap1 protein. Expression of total Nrf2, nuclear Nrf2, HO-1, NQO-1, and Keap1 was measured, with Western blot. Densitometric analysis was performed with Quantity One software 24 h later. Data are presented as means ± SD from 3 experiments. ∗∗ P
Figure Legend Snippet: L-malate (LM) increases expression of total Nrf2 and nuclear Nrf2 protein and HO-1, NQO-1 protein after MIRI and decreases expression of Keap1 protein. Expression of total Nrf2, nuclear Nrf2, HO-1, NQO-1, and Keap1 was measured, with Western blot. Densitometric analysis was performed with Quantity One software 24 h later. Data are presented as means ± SD from 3 experiments. ∗∗ P

Techniques Used: Expressing, Western Blot, Software

41) Product Images from "Nr2e1 Deficiency Augments Palmitate-Induced Oxidative Stress in Beta Cells"

Article Title: Nr2e1 Deficiency Augments Palmitate-Induced Oxidative Stress in Beta Cells

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2016/9648769

Effect of Nr2e1 on Nrf2 expression levels. (a) Relative expression level of Nfe2l2 mRNA in MIN6-shc, MIN6-sh2, and MIN6-sh3 cells. (b) Nrf2 protein levels. Results are means ± SD. ∗ P
Figure Legend Snippet: Effect of Nr2e1 on Nrf2 expression levels. (a) Relative expression level of Nfe2l2 mRNA in MIN6-shc, MIN6-sh2, and MIN6-sh3 cells. (b) Nrf2 protein levels. Results are means ± SD. ∗ P

Techniques Used: Expressing

42) Product Images from "Mechanisms of Triptolide-Induced Hepatotoxicity and Protective Effect of Combined Use of Isoliquiritigenin: Possible Roles of Nrf2 and Hepatic Transporters"

Article Title: Mechanisms of Triptolide-Induced Hepatotoxicity and Protective Effect of Combined Use of Isoliquiritigenin: Possible Roles of Nrf2 and Hepatic Transporters

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00226

ISL pretreatment induced expressions of (A) MRP2, (B) MRP4, and (C) P-gp to protect against TP-induced cytotoxicity. L02 cells were treated with 50 nM TP with or without 2.5, 5, and 7.5 μM ISL pretreatment. The tert -butylhydroquinone (15 μM) was used as an Nrf2 inducer. RT-PCR was used to analyze mRNAs and proteins by Western blot. Data were presented as means ± SE ( n = 3); ∗∗∗ P
Figure Legend Snippet: ISL pretreatment induced expressions of (A) MRP2, (B) MRP4, and (C) P-gp to protect against TP-induced cytotoxicity. L02 cells were treated with 50 nM TP with or without 2.5, 5, and 7.5 μM ISL pretreatment. The tert -butylhydroquinone (15 μM) was used as an Nrf2 inducer. RT-PCR was used to analyze mRNAs and proteins by Western blot. Data were presented as means ± SE ( n = 3); ∗∗∗ P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot

The proposed mechanism of TP-induced hepatotoxicity and ISL combined treatment protecting against TP-induced liver injury. ARE, antioxidant responsive element; BA, bile acid; BSEP, bile salt export pump; conj, conjugated; GSH, reduced glutathione; ISL, isoliquiritigenin; MRP, multidrug resistance protein; NQO1, NAD(P)H: quinine oxidoreductase 1; Nrf2, nuclear transcription factor E2-related factor 2; OATP, organic anion transporting polypeptide; P-gp, P-glycoprotein; sMaf, small Maf proteins; TP, triptolide.
Figure Legend Snippet: The proposed mechanism of TP-induced hepatotoxicity and ISL combined treatment protecting against TP-induced liver injury. ARE, antioxidant responsive element; BA, bile acid; BSEP, bile salt export pump; conj, conjugated; GSH, reduced glutathione; ISL, isoliquiritigenin; MRP, multidrug resistance protein; NQO1, NAD(P)H: quinine oxidoreductase 1; Nrf2, nuclear transcription factor E2-related factor 2; OATP, organic anion transporting polypeptide; P-gp, P-glycoprotein; sMaf, small Maf proteins; TP, triptolide.

Techniques Used:

Role of nuclear transcription factor E2-related factor 2 (Nrf2) in TP-induced cell death and ISL-induced protective effects. Effects of TP, ISL, or ISL + TP on the protein levels of (A) total, (B) cytoplasmic, and (C) nuclear Nrf2 and (D) NAD(P)H: quinine oxidoreductase 1 (NQO1) in L02 cells. Cells were treated with 50 nM TP with or without 2.5, 5, and 7.5 μM ISL pretreatment. The tBHQ (15 μM) was used as an Nrf2 inducer. Cell lysates were analyzed by Western blot. Role of Nrf2 in the protection against TP-induced cytotoxicity in L02 cells. Cells were transiently transfected with Nrf2-siRNA or Control-siRNA before TP, ISL, or ISL + TP treatment. (E) RT-PCR analysis of Nrf2 mRNA levels 48 h after the transfection. (F) Western blot analysis of nuclear Nrf2 protein levels 72 h after the transfection. (G) Cell viability determined by MTT assay. Data were presented as means ± SE ( n = 3); ∗∗ P
Figure Legend Snippet: Role of nuclear transcription factor E2-related factor 2 (Nrf2) in TP-induced cell death and ISL-induced protective effects. Effects of TP, ISL, or ISL + TP on the protein levels of (A) total, (B) cytoplasmic, and (C) nuclear Nrf2 and (D) NAD(P)H: quinine oxidoreductase 1 (NQO1) in L02 cells. Cells were treated with 50 nM TP with or without 2.5, 5, and 7.5 μM ISL pretreatment. The tBHQ (15 μM) was used as an Nrf2 inducer. Cell lysates were analyzed by Western blot. Role of Nrf2 in the protection against TP-induced cytotoxicity in L02 cells. Cells were transiently transfected with Nrf2-siRNA or Control-siRNA before TP, ISL, or ISL + TP treatment. (E) RT-PCR analysis of Nrf2 mRNA levels 48 h after the transfection. (F) Western blot analysis of nuclear Nrf2 protein levels 72 h after the transfection. (G) Cell viability determined by MTT assay. Data were presented as means ± SE ( n = 3); ∗∗ P

Techniques Used: Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, MTT Assay

Effects of TP, ISL, or ISL + TP on protein levels of (A) total, (B) cytoplasmic, and (C) nuclear Nrf2 and (D) Nqo1 in mouse livers. Cell lysates were analyzed by Western blot. Data were presented as means ± SE ( n = 5); ∗∗ P
Figure Legend Snippet: Effects of TP, ISL, or ISL + TP on protein levels of (A) total, (B) cytoplasmic, and (C) nuclear Nrf2 and (D) Nqo1 in mouse livers. Cell lysates were analyzed by Western blot. Data were presented as means ± SE ( n = 5); ∗∗ P

Techniques Used: Western Blot

43) Product Images from "Nuclear factor erythroid-derived factor 2-related factor 2 regulates transcription of CCAAT/enhancer-binding protein ? during adipogenesis"

Article Title: Nuclear factor erythroid-derived factor 2-related factor 2 regulates transcription of CCAAT/enhancer-binding protein ? during adipogenesis

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2011.10.453

Knockdown of Nrf2 suppresses C/EBPβ induction in 3T3-L1 cells. (A) Protein expression of Nrf2, C/EBPβ, C/EBPδ and PPARγ in the early stage of adipogenesis induced by DMI (upper panel) or DMIRI (lower panel). S, Scramble; N, Nrf2 - knockdown. (B) Transcription of C/EBPβ in the early stage of adipogenesis induced by DMI (upper panel) or DMIRI (lower panel). Scr, Scramble; Nrf2-KD, Nrf2 - knockdown. n = 3; *, p
Figure Legend Snippet: Knockdown of Nrf2 suppresses C/EBPβ induction in 3T3-L1 cells. (A) Protein expression of Nrf2, C/EBPβ, C/EBPδ and PPARγ in the early stage of adipogenesis induced by DMI (upper panel) or DMIRI (lower panel). S, Scramble; N, Nrf2 - knockdown. (B) Transcription of C/EBPβ in the early stage of adipogenesis induced by DMI (upper panel) or DMIRI (lower panel). Scr, Scramble; Nrf2-KD, Nrf2 - knockdown. n = 3; *, p

Techniques Used: Expressing

Expression of Nrf2-related transcription factors in the early stage of adipogenesis in 3T3-L1 cells induced by DMI (upper panel) or DMIRI (lower panel). Veh, vehicle (medium); S, Scramble; N, Nrf2 - knockdown.
Figure Legend Snippet: Expression of Nrf2-related transcription factors in the early stage of adipogenesis in 3T3-L1 cells induced by DMI (upper panel) or DMIRI (lower panel). Veh, vehicle (medium); S, Scramble; N, Nrf2 - knockdown.

Techniques Used: Expressing

Ablation of Nrf2 gene results in reduced adipogenesis in mouse WAT-derived preadipocytes. Preadipocytes isolated from WAT of Nrf2 -/- and Nrf2 +/+ mice were cultured to confluency and differentiated for 5 days using DMIRI protocol. Cells were then stained with ORO staining to visualize lipid accumulation (A) or used to measure the expression of adipogenic genes (B) . n = 3; * p
Figure Legend Snippet: Ablation of Nrf2 gene results in reduced adipogenesis in mouse WAT-derived preadipocytes. Preadipocytes isolated from WAT of Nrf2 -/- and Nrf2 +/+ mice were cultured to confluency and differentiated for 5 days using DMIRI protocol. Cells were then stained with ORO staining to visualize lipid accumulation (A) or used to measure the expression of adipogenic genes (B) . n = 3; * p

Techniques Used: Derivative Assay, Isolation, Mouse Assay, Cell Culture, Staining, Expressing

Effect of knockdown of Nrf2 and/or Keap1 on adipogenesis in 3T3-L1 preadipocytes. mRNA expression of Nrf2 (A) and Keap1 (B) in 3T3-L1 cells transduced with shRNA lentivirus targeted against mouse Nrf2 and/or Keap1 . Nrf2 -KD, Nrf2 -knockdown; Keap1 -KD, Keap1 - knockdown; Double-KD, Keap1 - and Nrf2 -double knockdown. n = 3; * p
Figure Legend Snippet: Effect of knockdown of Nrf2 and/or Keap1 on adipogenesis in 3T3-L1 preadipocytes. mRNA expression of Nrf2 (A) and Keap1 (B) in 3T3-L1 cells transduced with shRNA lentivirus targeted against mouse Nrf2 and/or Keap1 . Nrf2 -KD, Nrf2 -knockdown; Keap1 -KD, Keap1 - knockdown; Double-KD, Keap1 - and Nrf2 -double knockdown. n = 3; * p

Techniques Used: Expressing, Transduction, shRNA

Activation of Nrf2 occurs during adipogenesis in 3T3-L1 cells. (A) Nrf2 protein levels in nuclear fractions. Cells were treated with DMIRI for indicated time. S, Scramble; N, Nrf2 - knockdown; Veh, Vehicle (medium). (B) Quantification of Nrf2 level in (A). *, p
Figure Legend Snippet: Activation of Nrf2 occurs during adipogenesis in 3T3-L1 cells. (A) Nrf2 protein levels in nuclear fractions. Cells were treated with DMIRI for indicated time. S, Scramble; N, Nrf2 - knockdown; Veh, Vehicle (medium). (B) Quantification of Nrf2 level in (A). *, p

Techniques Used: Activation Assay

Expression of C/EBPβ is dependent on Nrf2 in 3T3-L1 cells. (A) Knockdown of Nrf2 suppresses expression of Cebpβ under basal and oxidative stressed conditions. Confluent cells were treated with iAs (10 μM) or tBHQ (25 μM) for 6 h. Scr, Scramble; Nrf2 -KD, Nrf2 -knockdown; n = 3; * p
Figure Legend Snippet: Expression of C/EBPβ is dependent on Nrf2 in 3T3-L1 cells. (A) Knockdown of Nrf2 suppresses expression of Cebpβ under basal and oxidative stressed conditions. Confluent cells were treated with iAs (10 μM) or tBHQ (25 μM) for 6 h. Scr, Scramble; Nrf2 -KD, Nrf2 -knockdown; n = 3; * p

Techniques Used: Expressing

44) Product Images from "Detoxification: A Novel Function of BRCA1 in Tumor Suppression?"

Article Title: Detoxification: A Novel Function of BRCA1 in Tumor Suppression?

Journal: Toxicological Sciences

doi: 10.1093/toxsci/kfr089

Effects of restoring ARNT or NRF2 on BaP-ind u ced DNA adducts in BRCA1 knockdown cells. (a) Cells were pretreated with siRNA (control vs. BRCA1), treated with BaP (0, 2.5, and 5μM), harvested, and confirmed by WB analysis using anti-BRCA1, anti-NRF2, and anti-ARNT antibodies. (b) Overexpression of ARNT in BRCA1 knockdown cells decreases the amount of adducts induced by BaP. Cells transfected with shRNA coding vector (pSuper-control vs. pSuper-BRCA1) for 48 h were transfected with GFP-tagged ARNT (vs. pEGFP as a empty vector) and then treated with [ 3 H]BaP for 24 h. (c) Restoring NRF2 reduced the amount of DNA adducts in BRCA1 knockdown cells. Cells were transfected with shRNA as in (a) and infected with adenovirus (Ad)-flag-NRF2 (vs. Ad-Null) and then [ 3 H]BaP-DNA adducts were measured. Student’s t -tests were applied for statistical significance; (*) and (**) indicate p
Figure Legend Snippet: Effects of restoring ARNT or NRF2 on BaP-ind u ced DNA adducts in BRCA1 knockdown cells. (a) Cells were pretreated with siRNA (control vs. BRCA1), treated with BaP (0, 2.5, and 5μM), harvested, and confirmed by WB analysis using anti-BRCA1, anti-NRF2, and anti-ARNT antibodies. (b) Overexpression of ARNT in BRCA1 knockdown cells decreases the amount of adducts induced by BaP. Cells transfected with shRNA coding vector (pSuper-control vs. pSuper-BRCA1) for 48 h were transfected with GFP-tagged ARNT (vs. pEGFP as a empty vector) and then treated with [ 3 H]BaP for 24 h. (c) Restoring NRF2 reduced the amount of DNA adducts in BRCA1 knockdown cells. Cells were transfected with shRNA as in (a) and infected with adenovirus (Ad)-flag-NRF2 (vs. Ad-Null) and then [ 3 H]BaP-DNA adducts were measured. Student’s t -tests were applied for statistical significance; (*) and (**) indicate p

Techniques Used: Western Blot, Over Expression, Transfection, shRNA, Plasmid Preparation, Infection

BRCA1 regulates the expression of endogenous UGT1A1, UGT1A9, and NRF2. Differential gene expression of UGT1A1, UGT1A9, or NRF2 in the presence and absence of endogenous BRCA1 was determined by semiquantitative RT-PCR and WB analysis. (a) BRCA1 levels affect the mRNA level of UGT1A1, UGT1A9, and NRF2 following BaP. Cells were pretreated with siRNA (control vs. BRCA1) for 72 h, treated with 5μM of BaP, and harvested at the indicated time points. The agarose gel image is a representative of three independent experiments. (b) Mean ± SE values from three independent semiquantitative RT-PCR assays were plotted to compare relative level of each transcript. (c) Cells were pretreated with siRNA (control vs. BRCA1) for 72 h, treated with 5μM of BaP (0, 2, 4, or 8 h), and harvested for WB analysis. (d) Mean ± SE values from three independent WB analysis were plotted to show relative intensity values of each band as determined by densitometry. Student’s t -tests were applied for statistical significance. Symbols (*) and (**) indicate p
Figure Legend Snippet: BRCA1 regulates the expression of endogenous UGT1A1, UGT1A9, and NRF2. Differential gene expression of UGT1A1, UGT1A9, or NRF2 in the presence and absence of endogenous BRCA1 was determined by semiquantitative RT-PCR and WB analysis. (a) BRCA1 levels affect the mRNA level of UGT1A1, UGT1A9, and NRF2 following BaP. Cells were pretreated with siRNA (control vs. BRCA1) for 72 h, treated with 5μM of BaP, and harvested at the indicated time points. The agarose gel image is a representative of three independent experiments. (b) Mean ± SE values from three independent semiquantitative RT-PCR assays were plotted to compare relative level of each transcript. (c) Cells were pretreated with siRNA (control vs. BRCA1) for 72 h, treated with 5μM of BaP (0, 2, 4, or 8 h), and harvested for WB analysis. (d) Mean ± SE values from three independent WB analysis were plotted to show relative intensity values of each band as determined by densitometry. Student’s t -tests were applied for statistical significance. Symbols (*) and (**) indicate p

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Agarose Gel Electrophoresis

BRCA1 is recruited to UGT1A1, UGT1A9, and NRF2 promoter/enhancer sequences. (a) ChIP assays were performed to determine the effect of BRCA1 knockdown on promoter occupancy by UGT1A1, UGT1A9, and NRF2. Fragmented genomic DNA from MCF-10A cells, which had been pretreated with siRNA for 72 h and then treated with 5μM BaP for 30 or 60 min, was immunoprecipitated by anti-BRCA1 and anti-ARNT antibodies. (b) RT-PCR of (a) results. Student’s t -tests were applied for statistical significance; ( # ) and ( ## ) indicate p
Figure Legend Snippet: BRCA1 is recruited to UGT1A1, UGT1A9, and NRF2 promoter/enhancer sequences. (a) ChIP assays were performed to determine the effect of BRCA1 knockdown on promoter occupancy by UGT1A1, UGT1A9, and NRF2. Fragmented genomic DNA from MCF-10A cells, which had been pretreated with siRNA for 72 h and then treated with 5μM BaP for 30 or 60 min, was immunoprecipitated by anti-BRCA1 and anti-ARNT antibodies. (b) RT-PCR of (a) results. Student’s t -tests were applied for statistical significance; ( # ) and ( ## ) indicate p

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

45) Product Images from "DJ-1 Knockout Augments Disease Severity and Shortens Survival in a Mouse Model of ALS"

Article Title: DJ-1 Knockout Augments Disease Severity and Shortens Survival in a Mouse Model of ALS

Journal: PLoS ONE

doi: 10.1371/journal.pone.0117190

Attenuated Nrf2 system in SOD DJ-1 KO mice. Mutated SOD1 increased the expression of Nrf2 and HO-1 while SOD1 DJ-1 KO demonstrated reduction. Nuclear factor erythroid-2 related factor 2 (Nrf2, A) and hemeoxygenase 1(HO-1, B) protein levels were determined in the spinal cord extracts by Western blot anlysis. Proteins levels were normalized to beta actin. Data is presented as averages ± SD. * p
Figure Legend Snippet: Attenuated Nrf2 system in SOD DJ-1 KO mice. Mutated SOD1 increased the expression of Nrf2 and HO-1 while SOD1 DJ-1 KO demonstrated reduction. Nuclear factor erythroid-2 related factor 2 (Nrf2, A) and hemeoxygenase 1(HO-1, B) protein levels were determined in the spinal cord extracts by Western blot anlysis. Proteins levels were normalized to beta actin. Data is presented as averages ± SD. * p

Techniques Used: Mouse Assay, Expressing, Western Blot

46) Product Images from "Vitamin A Supplementation Alleviates Extrahepatic Cholestasis Liver Injury through Nrf2 Activation"

Article Title: Vitamin A Supplementation Alleviates Extrahepatic Cholestasis Liver Injury through Nrf2 Activation

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2014/273692

Immunohistochemical staining of liver Nrf2 expression (original magnification: ×400; arrows indicate nucleus-positive cells). More Nrf2 was concentrated in the nuclei of the BDL group than in the SHAM group, and this concentration morphology was more apparent after treatment with vitamin A.
Figure Legend Snippet: Immunohistochemical staining of liver Nrf2 expression (original magnification: ×400; arrows indicate nucleus-positive cells). More Nrf2 was concentrated in the nuclei of the BDL group than in the SHAM group, and this concentration morphology was more apparent after treatment with vitamin A.

Techniques Used: Immunohistochemistry, Staining, Expressing, Concentration Assay

(a): Western blot results of Nrf2 expression in both the cytoplasm and nucleus from all three groups. Vitamin A significantly increased the level of Nrf2 in nucleus and decreased the level of Nrf2 in cytoplasm. PCNA and β -actin were used as protein loading control. (b): Gel shift assay showing Nrf2-ARE-binding activity was higher after BDL and much higher after treatment with vitamin A. SHAM group ( n = 10), BDL group ( n = 8), BDL + vitA group ( n = 9). Data are presented as mean ± SD. a P
Figure Legend Snippet: (a): Western blot results of Nrf2 expression in both the cytoplasm and nucleus from all three groups. Vitamin A significantly increased the level of Nrf2 in nucleus and decreased the level of Nrf2 in cytoplasm. PCNA and β -actin were used as protein loading control. (b): Gel shift assay showing Nrf2-ARE-binding activity was higher after BDL and much higher after treatment with vitamin A. SHAM group ( n = 10), BDL group ( n = 8), BDL + vitA group ( n = 9). Data are presented as mean ± SD. a P

Techniques Used: Western Blot, Expressing, Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay

47) Product Images from "Vitamin A Supplementation Alleviates Extrahepatic Cholestasis Liver Injury through Nrf2 Activation"

Article Title: Vitamin A Supplementation Alleviates Extrahepatic Cholestasis Liver Injury through Nrf2 Activation

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2014/273692

Immunohistochemical staining of liver Nrf2 expression (original magnification: ×400; arrows indicate nucleus-positive cells). More Nrf2 was concentrated in the nuclei of the BDL group than in the SHAM group, and this concentration morphology was more apparent after treatment with vitamin A.
Figure Legend Snippet: Immunohistochemical staining of liver Nrf2 expression (original magnification: ×400; arrows indicate nucleus-positive cells). More Nrf2 was concentrated in the nuclei of the BDL group than in the SHAM group, and this concentration morphology was more apparent after treatment with vitamin A.

Techniques Used: Immunohistochemistry, Staining, Expressing, Concentration Assay

(a): Western blot results of Nrf2 expression in both the cytoplasm and nucleus from all three groups. Vitamin A significantly increased the level of Nrf2 in nucleus and decreased the level of Nrf2 in cytoplasm. PCNA and β -actin were used as protein loading control. (b): Gel shift assay showing Nrf2-ARE-binding activity was higher after BDL and much higher after treatment with vitamin A. SHAM group ( n = 10), BDL group ( n = 8), BDL + vitA group ( n = 9). Data are presented as mean ± SD. a P
Figure Legend Snippet: (a): Western blot results of Nrf2 expression in both the cytoplasm and nucleus from all three groups. Vitamin A significantly increased the level of Nrf2 in nucleus and decreased the level of Nrf2 in cytoplasm. PCNA and β -actin were used as protein loading control. (b): Gel shift assay showing Nrf2-ARE-binding activity was higher after BDL and much higher after treatment with vitamin A. SHAM group ( n = 10), BDL group ( n = 8), BDL + vitA group ( n = 9). Data are presented as mean ± SD. a P

Techniques Used: Western Blot, Expressing, Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay

48) Product Images from "Mechanisms of Triptolide-Induced Hepatotoxicity and Protective Effect of Combined Use of Isoliquiritigenin: Possible Roles of Nrf2 and Hepatic Transporters"

Article Title: Mechanisms of Triptolide-Induced Hepatotoxicity and Protective Effect of Combined Use of Isoliquiritigenin: Possible Roles of Nrf2 and Hepatic Transporters

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00226

The proposed mechanism of TP-induced hepatotoxicity and ISL combined treatment protecting against TP-induced liver injury. ARE, antioxidant responsive element; BA, bile acid; BSEP, bile salt export pump; conj, conjugated; GSH, reduced glutathione; ISL, isoliquiritigenin; MRP, multidrug resistance protein; NQO1, NAD(P)H: quinine oxidoreductase 1; Nrf2, nuclear transcription factor E2-related factor 2; OATP, organic anion transporting polypeptide; P-gp, P-glycoprotein; sMaf, small Maf proteins; TP, triptolide.
Figure Legend Snippet: The proposed mechanism of TP-induced hepatotoxicity and ISL combined treatment protecting against TP-induced liver injury. ARE, antioxidant responsive element; BA, bile acid; BSEP, bile salt export pump; conj, conjugated; GSH, reduced glutathione; ISL, isoliquiritigenin; MRP, multidrug resistance protein; NQO1, NAD(P)H: quinine oxidoreductase 1; Nrf2, nuclear transcription factor E2-related factor 2; OATP, organic anion transporting polypeptide; P-gp, P-glycoprotein; sMaf, small Maf proteins; TP, triptolide.

Techniques Used:

Role of nuclear transcription factor E2-related factor 2 (Nrf2) in TP-induced cell death and ISL-induced protective effects. Effects of TP, ISL, or ISL + TP on the protein levels of (A) total, (B) cytoplasmic, and (C) nuclear Nrf2 and (D) NAD(P)H: quinine oxidoreductase 1 (NQO1) in L02 cells. Cells were treated with 50 nM TP with or without 2.5, 5, and 7.5 μM ISL pretreatment. The tBHQ (15 μM) was used as an Nrf2 inducer. Cell lysates were analyzed by Western blot. Role of Nrf2 in the protection against TP-induced cytotoxicity in L02 cells. Cells were transiently transfected with Nrf2-siRNA or Control-siRNA before TP, ISL, or ISL + TP treatment. (E) RT-PCR analysis of Nrf2 mRNA levels 48 h after the transfection. (F) Western blot analysis of nuclear Nrf2 protein levels 72 h after the transfection. (G) Cell viability determined by MTT assay. Data were presented as means ± SE ( n = 3); ∗∗ P
Figure Legend Snippet: Role of nuclear transcription factor E2-related factor 2 (Nrf2) in TP-induced cell death and ISL-induced protective effects. Effects of TP, ISL, or ISL + TP on the protein levels of (A) total, (B) cytoplasmic, and (C) nuclear Nrf2 and (D) NAD(P)H: quinine oxidoreductase 1 (NQO1) in L02 cells. Cells were treated with 50 nM TP with or without 2.5, 5, and 7.5 μM ISL pretreatment. The tBHQ (15 μM) was used as an Nrf2 inducer. Cell lysates were analyzed by Western blot. Role of Nrf2 in the protection against TP-induced cytotoxicity in L02 cells. Cells were transiently transfected with Nrf2-siRNA or Control-siRNA before TP, ISL, or ISL + TP treatment. (E) RT-PCR analysis of Nrf2 mRNA levels 48 h after the transfection. (F) Western blot analysis of nuclear Nrf2 protein levels 72 h after the transfection. (G) Cell viability determined by MTT assay. Data were presented as means ± SE ( n = 3); ∗∗ P

Techniques Used: Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, MTT Assay

Effects of TP, ISL, or ISL + TP on protein levels of (A) total, (B) cytoplasmic, and (C) nuclear Nrf2 and (D) Nqo1 in mouse livers. Cell lysates were analyzed by Western blot. Data were presented as means ± SE ( n = 5); ∗∗ P
Figure Legend Snippet: Effects of TP, ISL, or ISL + TP on protein levels of (A) total, (B) cytoplasmic, and (C) nuclear Nrf2 and (D) Nqo1 in mouse livers. Cell lysates were analyzed by Western blot. Data were presented as means ± SE ( n = 5); ∗∗ P

Techniques Used: Western Blot

49) Product Images from "Histone deacetylase inhibition activates transcription factor Nrf2 and protects against cerebral ischemic damage"

Article Title: Histone deacetylase inhibition activates transcription factor Nrf2 and protects against cerebral ischemic damage

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2011.12.006

TSA activates Nrf2 and enhances Nrf2–ARE binding. (A) RAW 264.7 cells were treated with TSA (30 ng/mL), EGb 761 (100 μg/mL), or TSA+EGb 761 for 16 h. Coimmuno-precipitation showed that antibodies to Keap1 and Nrf2 precipitated significantly less of the other protein after treatment with one or both drugs, indicating a reduction in interaction between Nrf2 and Keap1. Total Keap1 and total Nrf2 were used as protein loading controls. (B) Nrf2 protein expression was assessed in nuclear and cytosolic fractions. Nrf2 expression was unchanged in the cytosolic fraction, but it was significantly increased in the nuclear fraction after TSA treatment and cotreatment with TSA and EGb 761. (C) Based on EMSA, Nrf2–ARE-binding activity was enhanced after TSA treatment and substantially increased after treatment with TSA+EGb 761. Competition with excess cold (nonradioactive) probes markedly suppressed Nrf2–ARE binding, but mutant probes were unable to disrupt this binding. (D) In a chromatin immunoprecipitation assay, treatment with TSA alone or with EGb 761 induced Nrf2 association with HO1 enhancers at both −4 K and −10 K; Nrf2 did not bind to HO1 proximal promoters with non-Nrf2-binding sites. GAPDH was a loading control. (E) The luciferase assay showed that TSA or TSA+EGb 761 induced luciferase activity; TSA+EGb 761 increased transcription more than either drug individually. * P
Figure Legend Snippet: TSA activates Nrf2 and enhances Nrf2–ARE binding. (A) RAW 264.7 cells were treated with TSA (30 ng/mL), EGb 761 (100 μg/mL), or TSA+EGb 761 for 16 h. Coimmuno-precipitation showed that antibodies to Keap1 and Nrf2 precipitated significantly less of the other protein after treatment with one or both drugs, indicating a reduction in interaction between Nrf2 and Keap1. Total Keap1 and total Nrf2 were used as protein loading controls. (B) Nrf2 protein expression was assessed in nuclear and cytosolic fractions. Nrf2 expression was unchanged in the cytosolic fraction, but it was significantly increased in the nuclear fraction after TSA treatment and cotreatment with TSA and EGb 761. (C) Based on EMSA, Nrf2–ARE-binding activity was enhanced after TSA treatment and substantially increased after treatment with TSA+EGb 761. Competition with excess cold (nonradioactive) probes markedly suppressed Nrf2–ARE binding, but mutant probes were unable to disrupt this binding. (D) In a chromatin immunoprecipitation assay, treatment with TSA alone or with EGb 761 induced Nrf2 association with HO1 enhancers at both −4 K and −10 K; Nrf2 did not bind to HO1 proximal promoters with non-Nrf2-binding sites. GAPDH was a loading control. (E) The luciferase assay showed that TSA or TSA+EGb 761 induced luciferase activity; TSA+EGb 761 increased transcription more than either drug individually. * P

Techniques Used: Binding Assay, Expressing, Activity Assay, Mutagenesis, Chromatin Immunoprecipitation, Luciferase

TSA and other HDAC inhibitors suppress Nrf2 inhibitor Keap1 expression. Different concentrations of TSA (10, 30, and 100 ng/mL), EGb 761 (3, 10, 30, and 100 μg/mL), or both were applied to mouse RAW 264.7 cells for 16 h. Then Keap1 protein levels were measured by Western blot analysis. (A) TSA and TSA+EGb 761, but not EGb 761 alone, decreased Keap1 expression at all doses tested. (B) TSA (30 ng/mL) significantly reduced Keap1 protein level at 16 h. (C) TSA (30 ng/mL) and TSA+EGb 761 (100 μg/mL) reduced Keap1 mRNA levels as measured by RT-PCR. (D, E) Commonly used HDAC inhibitors sodium butyrate (NaB, 5 mg/L) and MS275 (10 μmol/L) decreased Keap1 expression in the presence or absence of EGb 761 after 16 h of treatment. (F) Treatment with TSA (30 ng/mL) or TSA+EGb 761 (100 μg/mL) produced a trend toward Nrf2 acetylation. Ac-Nrf2, acetylated Nrf2; T-Nrf2, total Nrf2.
Figure Legend Snippet: TSA and other HDAC inhibitors suppress Nrf2 inhibitor Keap1 expression. Different concentrations of TSA (10, 30, and 100 ng/mL), EGb 761 (3, 10, 30, and 100 μg/mL), or both were applied to mouse RAW 264.7 cells for 16 h. Then Keap1 protein levels were measured by Western blot analysis. (A) TSA and TSA+EGb 761, but not EGb 761 alone, decreased Keap1 expression at all doses tested. (B) TSA (30 ng/mL) significantly reduced Keap1 protein level at 16 h. (C) TSA (30 ng/mL) and TSA+EGb 761 (100 μg/mL) reduced Keap1 mRNA levels as measured by RT-PCR. (D, E) Commonly used HDAC inhibitors sodium butyrate (NaB, 5 mg/L) and MS275 (10 μmol/L) decreased Keap1 expression in the presence or absence of EGb 761 after 16 h of treatment. (F) Treatment with TSA (30 ng/mL) or TSA+EGb 761 (100 μg/mL) produced a trend toward Nrf2 acetylation. Ac-Nrf2, acetylated Nrf2; T-Nrf2, total Nrf2.

Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Produced

TSA upregulates the expression of proteins downstream of Nrf2. TSA (30 ng/mL) and EGb 761 (100 μg/mL) increased HO1 expression in mouse RAW 264.7 cells (A) and in primary neuronal cells (B). TSA further enhanced EGb 761-induced HO1 expression (A, B). Additionally, immunoblotting showed that the expression of two other Nrf2-regulated proteins, NQO1 and GCLC, was elevated by TSA treatment in cultured neuronal cells (C) and in brain tissue (D). TSA further increased NQO1 and GCLC expression induced by EGb 761 in neurons (C). Actin was used as a loading control.
Figure Legend Snippet: TSA upregulates the expression of proteins downstream of Nrf2. TSA (30 ng/mL) and EGb 761 (100 μg/mL) increased HO1 expression in mouse RAW 264.7 cells (A) and in primary neuronal cells (B). TSA further enhanced EGb 761-induced HO1 expression (A, B). Additionally, immunoblotting showed that the expression of two other Nrf2-regulated proteins, NQO1 and GCLC, was elevated by TSA treatment in cultured neuronal cells (C) and in brain tissue (D). TSA further increased NQO1 and GCLC expression induced by EGb 761 in neurons (C). Actin was used as a loading control.

Techniques Used: Expressing, Cell Culture

TSA protection against cerebral ischemia is absent in Nrf2-deficient mice. Nrf2 −/− mice had increased infarct volume compared to WT counterparts in the absence of TSA (A), and also had more neurological deficits than WT mice, but no statistical significance (C). TSA treatment (1 mg/kg) had no effect on cortical infarct volume or neurologic deficit scores in Nrf2 −/− mice compared to vehicle-treated controls (B and D); n =10 per group.
Figure Legend Snippet: TSA protection against cerebral ischemia is absent in Nrf2-deficient mice. Nrf2 −/− mice had increased infarct volume compared to WT counterparts in the absence of TSA (A), and also had more neurological deficits than WT mice, but no statistical significance (C). TSA treatment (1 mg/kg) had no effect on cortical infarct volume or neurologic deficit scores in Nrf2 −/− mice compared to vehicle-treated controls (B and D); n =10 per group.

Techniques Used: Mouse Assay

50) Product Images from "A Novel Approach to Screening for New Neuroprotective Compounds for the Treatment of Stroke"

Article Title: A Novel Approach to Screening for New Neuroprotective Compounds for the Treatment of Stroke

Journal: Brain research

doi: 10.1016/j.brainres.2007.07.061

Flavonoids Induce the Expression of Nrf2, the ARE-specific transcription factor, and HO-1, a phase II detoxification protein
Figure Legend Snippet: Flavonoids Induce the Expression of Nrf2, the ARE-specific transcription factor, and HO-1, a phase II detoxification protein

Techniques Used: Expressing

51) Product Images from "Vitamin E prevents NRF2-suppression by allergen in asthmatic alveolar macrophages in vivo"

Article Title: Vitamin E prevents NRF2-suppression by allergen in asthmatic alveolar macrophages in vivo

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2011.04.040

A. Ex vivo effect of sulforaphane on NRF2 protein expression in alveolar macrophages from an atopic asthmatic at baseline and at 24 h after allergen challenge in vivo . B. Ex vivo effect of CDDO on DNA binding activity of NRF2 in alveolar macrophages from
Figure Legend Snippet: A. Ex vivo effect of sulforaphane on NRF2 protein expression in alveolar macrophages from an atopic asthmatic at baseline and at 24 h after allergen challenge in vivo . B. Ex vivo effect of CDDO on DNA binding activity of NRF2 in alveolar macrophages from

Techniques Used: Ex Vivo, Expressing, In Vivo, Binding Assay, Activity Assay

DNA binding activity of NRF2 in alveolar macrophages from the control lung segment (Baseline) and at 24 h after allergen challenge in atopic asthmatics (Allergen), and 5 healthy subjects (non-atopic and non-asthmatic) (Healthy subjects) in vivo . Segmental
Figure Legend Snippet: DNA binding activity of NRF2 in alveolar macrophages from the control lung segment (Baseline) and at 24 h after allergen challenge in atopic asthmatics (Allergen), and 5 healthy subjects (non-atopic and non-asthmatic) (Healthy subjects) in vivo . Segmental

Techniques Used: Binding Assay, Activity Assay, In Vivo

RT-PCR of the NRF2 gene in alveolar macrophages from the control lung segment (Baseline) and at 24 h after allergen challenge (Allergen) in atopic asthmatics in vivo . NRF2 expression is normalized to expression of GADPH. Red, green, and purple, colors
Figure Legend Snippet: RT-PCR of the NRF2 gene in alveolar macrophages from the control lung segment (Baseline) and at 24 h after allergen challenge (Allergen) in atopic asthmatics in vivo . NRF2 expression is normalized to expression of GADPH. Red, green, and purple, colors

Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vivo, Expressing

A. Comparison of NRF2 protein expression in alveolar macrophages from the control lung segment and at 24 h after allergen challenge, before (Bas and All, respectively) and after supplementation of vitamin E (Bas-E and All-E, respectively) in atopic asthmatics
Figure Legend Snippet: A. Comparison of NRF2 protein expression in alveolar macrophages from the control lung segment and at 24 h after allergen challenge, before (Bas and All, respectively) and after supplementation of vitamin E (Bas-E and All-E, respectively) in atopic asthmatics

Techniques Used: Expressing

52) Product Images from "Nrf2 induces cisplatin resistance via suppressing the iron export related gene SLC40A1 in ovarian cancer cells"

Article Title: Nrf2 induces cisplatin resistance via suppressing the iron export related gene SLC40A1 in ovarian cancer cells

Journal: Oncotarget

doi: 10.18632/oncotarget.19548

Positive feedback reaction of SLC40A1 on Nrf2 (A and C) Western blot was employed to determine the protein expression of Nrf2/NQO1, when SLC40A1 expression was inhibited with specific siRNA (50nM) in A2780 (A) and COC1 (C) ovarian cancer cells. (B and D) The protein expression of Nrf2/NQO1were detected by western blot, when the expression of SLC40A1 was enhanced through PCDH-SLC40A1 transfection in A2780CP (B) and COC1/DDP (D) ovarian cancer cells.
Figure Legend Snippet: Positive feedback reaction of SLC40A1 on Nrf2 (A and C) Western blot was employed to determine the protein expression of Nrf2/NQO1, when SLC40A1 expression was inhibited with specific siRNA (50nM) in A2780 (A) and COC1 (C) ovarian cancer cells. (B and D) The protein expression of Nrf2/NQO1were detected by western blot, when the expression of SLC40A1 was enhanced through PCDH-SLC40A1 transfection in A2780CP (B) and COC1/DDP (D) ovarian cancer cells.

Techniques Used: Western Blot, Expressing, Transfection

Schematic: SLC40A1 promotes iron export and prevents Nrf2 dependent cisplatin resistance in ovarian cancer Nrf2 is activated and transported into cell nucleus after cisplatin treatment in cisplatin-resistant ovarian cancer cells. Increased Nrf2 in cell nucleus combines to the binding sites (+336∼+432,-1220∼-1320) to transcriptionally inhibit the expression of SLC40A1, thus resulting in cisplatin resistance. However, SLC40A1 also has a positive feedback interaction with Nrf2. Moreover, as a novel iron exporter, abnormal expression of SLC40A1 is associated with cisplatin resistance and increased intracellular iron. While increased intracellular iron induced by Fecl3 results in cisplatin resistance. Inversely, desferal, an iron chelator, overcomes cisplatin resistance by reducing intracellular free iron. Brusatol enhances the antitumor effect of desferal by inhibiting expression of Nrf2 in cisplatin-resistant ovarian cancers.
Figure Legend Snippet: Schematic: SLC40A1 promotes iron export and prevents Nrf2 dependent cisplatin resistance in ovarian cancer Nrf2 is activated and transported into cell nucleus after cisplatin treatment in cisplatin-resistant ovarian cancer cells. Increased Nrf2 in cell nucleus combines to the binding sites (+336∼+432,-1220∼-1320) to transcriptionally inhibit the expression of SLC40A1, thus resulting in cisplatin resistance. However, SLC40A1 also has a positive feedback interaction with Nrf2. Moreover, as a novel iron exporter, abnormal expression of SLC40A1 is associated with cisplatin resistance and increased intracellular iron. While increased intracellular iron induced by Fecl3 results in cisplatin resistance. Inversely, desferal, an iron chelator, overcomes cisplatin resistance by reducing intracellular free iron. Brusatol enhances the antitumor effect of desferal by inhibiting expression of Nrf2 in cisplatin-resistant ovarian cancers.

Techniques Used: Binding Assay, Expressing

The inhibition of Nrf2 on SLC40A1 in ovarian cancer cells (A) The protein expressions of SLC40A1 were detected, when A2780 or A2780CP was treated with PCDH-Nrf2 vector or Nrf2 shRNA for 72h. (B) The protein expressions of SLC40A1 were determined, when A2780 was treated with Nrf2 activator (TBHQ, 50umol/l) for 16h, or A2780CP was treated with Nrf2 inhibitor (brusatol, 50nmol/l) for 3h. (C) The protein expressions of SLC40A1 were detected, when COC1 or COC1/DDP was treated with PCDH-Nrf2 vector or Nrf2 shRNA for 72h. (D) The protein expressions of SLC40A1 were determined, when COC1 was treated with Nrf2 activator (TBHQ, 50umol/l) for 16h, or COC1/DDP was treated with Nrf2 inhibitor (brusatol, 50nmol/l) for 3h.
Figure Legend Snippet: The inhibition of Nrf2 on SLC40A1 in ovarian cancer cells (A) The protein expressions of SLC40A1 were detected, when A2780 or A2780CP was treated with PCDH-Nrf2 vector or Nrf2 shRNA for 72h. (B) The protein expressions of SLC40A1 were determined, when A2780 was treated with Nrf2 activator (TBHQ, 50umol/l) for 16h, or A2780CP was treated with Nrf2 inhibitor (brusatol, 50nmol/l) for 3h. (C) The protein expressions of SLC40A1 were detected, when COC1 or COC1/DDP was treated with PCDH-Nrf2 vector or Nrf2 shRNA for 72h. (D) The protein expressions of SLC40A1 were determined, when COC1 was treated with Nrf2 activator (TBHQ, 50umol/l) for 16h, or COC1/DDP was treated with Nrf2 inhibitor (brusatol, 50nmol/l) for 3h.

Techniques Used: Inhibition, Plasmid Preparation, shRNA

SLC40A1 inverts cisplatin resistance induced by Nrf2 (A and C) A2780 (A) or COC1 (C) was transfected with control vector (2ug), PCDH-Nrf2 plasmid (2ug), and PCDH/SLC40A1 (2ug) for 48 h. Then, they were treated with different levels of cisplatin for 24h to detect cell viability. (B and D) A2780CP (B) or COC1/DDP (D) was transfected with control siRNA (50nM), Nrf2 shRNA (2ug), and SLC40A1 siRNA (50nM) for 48 h. Then, they were treated with different levels of cisplatin for 24h to detect cell viability. * P
Figure Legend Snippet: SLC40A1 inverts cisplatin resistance induced by Nrf2 (A and C) A2780 (A) or COC1 (C) was transfected with control vector (2ug), PCDH-Nrf2 plasmid (2ug), and PCDH/SLC40A1 (2ug) for 48 h. Then, they were treated with different levels of cisplatin for 24h to detect cell viability. (B and D) A2780CP (B) or COC1/DDP (D) was transfected with control siRNA (50nM), Nrf2 shRNA (2ug), and SLC40A1 siRNA (50nM) for 48 h. Then, they were treated with different levels of cisplatin for 24h to detect cell viability. * P

Techniques Used: Transfection, Plasmid Preparation, shRNA

Nrf2 transcriptionally suppresses the expression of SLC40A1 (A) 5 * 10 3 HEK-293T cells were transfected with different concentrations of PCDH-Nrf2 plasmid and pGL3-SLC40A1 reporter gene plasmid for 48 h. Then the promotor activity of SLC40A1 was determined by dual-luciferase reporter assay. (B) Left panel, different length of SLC40A1 promotor sequences were established according to putative binding sites and were linked to the reporter gene vector pGL3. Right panel, the relative luciferase activities among different SLC40A1 promotor sequences were analyzed by luciferase assay. (C) The interaction of Nrf2 with the SLC40A1 promotor was assayed by ChIP analysis. Numerous primers were designed according to the putative binding sites to detect the possible binding site. The reagent with no DNA was used as blank control. Total DNA was extracted and used as input. The normal mouse IgG was employed as the negative control. The GAPDH antibody was used as the positive control. (D) Sequence analysis indicated that there were two Nrf2 binding sites in the promotor of SLC40A1 (+336∼ +432, -1320∼ -1220). * P
Figure Legend Snippet: Nrf2 transcriptionally suppresses the expression of SLC40A1 (A) 5 * 10 3 HEK-293T cells were transfected with different concentrations of PCDH-Nrf2 plasmid and pGL3-SLC40A1 reporter gene plasmid for 48 h. Then the promotor activity of SLC40A1 was determined by dual-luciferase reporter assay. (B) Left panel, different length of SLC40A1 promotor sequences were established according to putative binding sites and were linked to the reporter gene vector pGL3. Right panel, the relative luciferase activities among different SLC40A1 promotor sequences were analyzed by luciferase assay. (C) The interaction of Nrf2 with the SLC40A1 promotor was assayed by ChIP analysis. Numerous primers were designed according to the putative binding sites to detect the possible binding site. The reagent with no DNA was used as blank control. Total DNA was extracted and used as input. The normal mouse IgG was employed as the negative control. The GAPDH antibody was used as the positive control. (D) Sequence analysis indicated that there were two Nrf2 binding sites in the promotor of SLC40A1 (+336∼ +432, -1320∼ -1220). * P

Techniques Used: Expressing, Transfection, Plasmid Preparation, Activity Assay, Luciferase, Reporter Assay, Binding Assay, Chromatin Immunoprecipitation, Negative Control, Positive Control, Sequencing

SLC40A1 and Nrf2 expression in cisplatin-sensitive ovarian cancer cells and cisplatin-resistant ovarian cancer cells (A) The mRNA expressions of Nrf2 and SLC40A1 in cisplatin-sensitive ovarian cancer cells (blue bar) and their derived cisplatin-resistant ovarian cancer cells (red bar) were determined by qRT-PCR. (B) Western blot was employed to detect the protein expression of Nrf2 and SLC40A1 in A2780 and its derived A2780CP ovarian cancer cells. (C) The protein expression of Nrf2 and SLC40A1 in COC1 and its derived COC1/DDP ovarian cancer cells were also determined through Western blot, * P
Figure Legend Snippet: SLC40A1 and Nrf2 expression in cisplatin-sensitive ovarian cancer cells and cisplatin-resistant ovarian cancer cells (A) The mRNA expressions of Nrf2 and SLC40A1 in cisplatin-sensitive ovarian cancer cells (blue bar) and their derived cisplatin-resistant ovarian cancer cells (red bar) were determined by qRT-PCR. (B) Western blot was employed to detect the protein expression of Nrf2 and SLC40A1 in A2780 and its derived A2780CP ovarian cancer cells. (C) The protein expression of Nrf2 and SLC40A1 in COC1 and its derived COC1/DDP ovarian cancer cells were also determined through Western blot, * P

Techniques Used: Expressing, Derivative Assay, Quantitative RT-PCR, Western Blot

53) Product Images from "Frataxin Deficiency Leads to Defects in Expression of Antioxidants and Nrf2 Expression in Dorsal Root Ganglia of the Friedreich's Ataxia YG8R Mouse Model"

Article Title: Frataxin Deficiency Leads to Defects in Expression of Antioxidants and Nrf2 Expression in Dorsal Root Ganglia of the Friedreich's Ataxia YG8R Mouse Model

Journal: Antioxidants & Redox Signaling

doi: 10.1089/ars.2012.4537

Nrf2 and downstream target proteins are decreased in YG8R DRGs. (A) Representative immunoblots of Nrf2, Hmox, Nqo1, and Sod2 in the YG8R DRGs. Each lane represents an individual mouse. (B) Densitometry of blots shown in A, normalized to tubulin. Each
Figure Legend Snippet: Nrf2 and downstream target proteins are decreased in YG8R DRGs. (A) Representative immunoblots of Nrf2, Hmox, Nqo1, and Sod2 in the YG8R DRGs. Each lane represents an individual mouse. (B) Densitometry of blots shown in A, normalized to tubulin. Each

Techniques Used: Western Blot

Transcripts encoding Nrf2 and downstream targets are decreased in YG8R DRGs and correlated with frataxin levels. (A–H) Correlation analysis of the frataxin transcript levels with Nrf2 (A) , Cat (B) Gclc (C) , Gclm (D) , Hmox (E) , Sod1 (F) , Sod2
Figure Legend Snippet: Transcripts encoding Nrf2 and downstream targets are decreased in YG8R DRGs and correlated with frataxin levels. (A–H) Correlation analysis of the frataxin transcript levels with Nrf2 (A) , Cat (B) Gclc (C) , Gclm (D) , Hmox (E) , Sod1 (F) , Sod2

Techniques Used:

Nrf2 is decreased in frataxin-deficient cells. (A) Nrf2 protein decreased in HeLa and T265 frataxin-depleted cells. (B) No Nrf2 nuclear translocation defect in frataxin-deficient cells. Nuclear extraction and immunopurified anti-Nrf2 antibody demonstrate
Figure Legend Snippet: Nrf2 is decreased in frataxin-deficient cells. (A) Nrf2 protein decreased in HeLa and T265 frataxin-depleted cells. (B) No Nrf2 nuclear translocation defect in frataxin-deficient cells. Nuclear extraction and immunopurified anti-Nrf2 antibody demonstrate

Techniques Used: Translocation Assay

54) Product Images from "Effect of DJ-1 on the neuroprotection of astrocytes subjected to cerebral ischemia/reperfusion injury"

Article Title: Effect of DJ-1 on the neuroprotection of astrocytes subjected to cerebral ischemia/reperfusion injury

Journal: Journal of Molecular Medicine (Berlin, Germany)

doi: 10.1007/s00109-018-1719-5

Effects of DJ-1 on expression of Nrf2. a After OGD for 5 h followed by 24 h of reoxygenation, astrocytes were harvested. Western blot for Nrf2 and CRIF1 in astrocytes. b After 24 h of reperfusion, brains were collected. Western blot for Nrf2 and CRIF1 in rats. c , d Ratios of Nrf2 and CRIF1 relative to β-actin in astrocytes and in rats, respectively. e Immunocytochemistry assays to measure total Nrf2 and nuclear Nrf2 in astrocytes. Fluorescence microscopy was used to assess expression. Nrf2 expression in astrocytes is indicated by red fluorescence. Cell nuclei were stained with DAPI. Original magnification, 200×. f Semi-quantitation to determine total number of Nrf2-positive cells and number of nuclear Nrf2-positive cells in astrocytes. Values are mean ± SEM. # p
Figure Legend Snippet: Effects of DJ-1 on expression of Nrf2. a After OGD for 5 h followed by 24 h of reoxygenation, astrocytes were harvested. Western blot for Nrf2 and CRIF1 in astrocytes. b After 24 h of reperfusion, brains were collected. Western blot for Nrf2 and CRIF1 in rats. c , d Ratios of Nrf2 and CRIF1 relative to β-actin in astrocytes and in rats, respectively. e Immunocytochemistry assays to measure total Nrf2 and nuclear Nrf2 in astrocytes. Fluorescence microscopy was used to assess expression. Nrf2 expression in astrocytes is indicated by red fluorescence. Cell nuclei were stained with DAPI. Original magnification, 200×. f Semi-quantitation to determine total number of Nrf2-positive cells and number of nuclear Nrf2-positive cells in astrocytes. Values are mean ± SEM. # p

Techniques Used: Expressing, Western Blot, Immunocytochemistry, Fluorescence, Microscopy, Staining, Quantitation Assay

DJ-1 regulates Nrf2/ARE binding activity and Nrf2/ARE-driven gene expression. After OGD for 5 h followed by 24 h of reoxygenation, astrocytes were harvested. And after 24 h of reperfusion, brains were collected. a EMSA analysis of Nrf2/ARE binding. b Semiquantitative analysis of Nrf2/ARE binding. CK, 100x, (+) and (−) indicate controls. c Western blot for GCLM, GCLC, and GSS in astrocytes. d Western blot for GCLM, GCLC, and GSS in rats. e , f Ratios of GCLM, GCLC, and GSS relative to β-actin in astrocytes and in rats, respectively. Values are mean ± SEM. # p
Figure Legend Snippet: DJ-1 regulates Nrf2/ARE binding activity and Nrf2/ARE-driven gene expression. After OGD for 5 h followed by 24 h of reoxygenation, astrocytes were harvested. And after 24 h of reperfusion, brains were collected. a EMSA analysis of Nrf2/ARE binding. b Semiquantitative analysis of Nrf2/ARE binding. CK, 100x, (+) and (−) indicate controls. c Western blot for GCLM, GCLC, and GSS in astrocytes. d Western blot for GCLM, GCLC, and GSS in rats. e , f Ratios of GCLM, GCLC, and GSS relative to β-actin in astrocytes and in rats, respectively. Values are mean ± SEM. # p

Techniques Used: Binding Assay, Activity Assay, Expressing, Western Blot

Mechanisms of action of DJ-1 in astrocytes. After oxidative stress induced by cerebral ischemia and reperfusion, DJ-1 is expressed in immunoreactive astrocytes. DJ-1 facilitates Nrf2 translocation to the nucleus by preventing binding with Keap1. Nrf2 binds to ARE and upregulates expression of GCLC, GCLM, and GSS, which regulate synthesis of GSH
Figure Legend Snippet: Mechanisms of action of DJ-1 in astrocytes. After oxidative stress induced by cerebral ischemia and reperfusion, DJ-1 is expressed in immunoreactive astrocytes. DJ-1 facilitates Nrf2 translocation to the nucleus by preventing binding with Keap1. Nrf2 binds to ARE and upregulates expression of GCLC, GCLM, and GSS, which regulate synthesis of GSH

Techniques Used: Translocation Assay, Binding Assay, Expressing

55) Product Images from "Transforming Growth Factor-? and Nuclear Factor E2-related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells"

Article Title: Transforming Growth Factor-? and Nuclear Factor E2-related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells

Journal: American Journal of Respiratory and Critical Care Medicine

doi: 10.1164/rccm.201011-1780OC

( A and B ) Confluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours and then treated with transforming growth factor (TGF)-β (1 ng/ml) for 0.5–24 hours ( A ), or TGF-β (0.25–1 ng/ml) for 24 hours ( B ). Heme oxygenase (HO)-1 and NAD(P)H:quinone oxidoreductase (NQO1) mRNA was determined by real-time polymerase chain reaction (PCR) and normalized to 18S rRNA expression. ( C and D ) Confluent ASMCs were transfected with antioxidant response elements (ARE)–driven luciferase reporter vector for 18 hours, serum-deprived for 6 hours, and finally treated with TGF-β (0.25–1 ng/ml) for 20 hours ( C ) or pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour and then treated with TGF-β (0.25 ng/ml) for 20 hours ( D ). ARE-driven transcriptional activity was determined by measuring firefly luciferase activity and normalizing to Renilla luciferase activity. ( E and F ) Confluent ASMCs were serum-deprived for 24 hours and then treated with TGF-β (0.25–1 ng/ml) for 20 hours. Nuclear factor E2-related factor 2 (Nrf2) expression was determined in whole-cell extracts by Western blotting and normalized to β-actin expression ( E ). Nrf2-ARE binding was determined in nuclear extracts by an ELISA-based TransAM assay ( F ). Bars represent mean ± SEM of three ASMC ( A and F ), five ASMC ( B ), three to six ASMC ( C ), four ASMC ( D ), and four ASMC donors ( E ). * P
Figure Legend Snippet: ( A and B ) Confluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours and then treated with transforming growth factor (TGF)-β (1 ng/ml) for 0.5–24 hours ( A ), or TGF-β (0.25–1 ng/ml) for 24 hours ( B ). Heme oxygenase (HO)-1 and NAD(P)H:quinone oxidoreductase (NQO1) mRNA was determined by real-time polymerase chain reaction (PCR) and normalized to 18S rRNA expression. ( C and D ) Confluent ASMCs were transfected with antioxidant response elements (ARE)–driven luciferase reporter vector for 18 hours, serum-deprived for 6 hours, and finally treated with TGF-β (0.25–1 ng/ml) for 20 hours ( C ) or pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour and then treated with TGF-β (0.25 ng/ml) for 20 hours ( D ). ARE-driven transcriptional activity was determined by measuring firefly luciferase activity and normalizing to Renilla luciferase activity. ( E and F ) Confluent ASMCs were serum-deprived for 24 hours and then treated with TGF-β (0.25–1 ng/ml) for 20 hours. Nuclear factor E2-related factor 2 (Nrf2) expression was determined in whole-cell extracts by Western blotting and normalized to β-actin expression ( E ). Nrf2-ARE binding was determined in nuclear extracts by an ELISA-based TransAM assay ( F ). Bars represent mean ± SEM of three ASMC ( A and F ), five ASMC ( B ), three to six ASMC ( C ), four ASMC ( D ), and four ASMC donors ( E ). * P

Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

( A and B ) Semiconfluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours, pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour, and then incubated with medium containing 2.5% fetal bovine serum (FBS) or 2.5% FBS and transforming growth factor (TGF)-β (0.25 ng/ml) for 72 hours ( A ). Alternatively, ASMCs were incubated with adenoviral vectors expressing GFP (Ad-GFP) and wild-type nuclear factor E2-related factor 2 (Ad-Nrf2) (multiplicity of infection 250) for 18 hours, serum-deprived for 6 hours, and then incubated with medium containing 2.5% FBS or 2.5% FBS and TGF-β (0.25 ng/ml) for 72 hours ( B ). DNA synthesis was determined by measuring bromodeoxyuridine (BrdU) incorporation. ( C–F ) Confluent ASMCs were serum-deprived for 24 hours, pretreated with vehicle control or sulforaphane (2–4 μM) for 1 hour, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours ( C and D ). Alternatively, ASMCs were incubated with Ad-GFP or Ad-Nrf2 (MOI 250) for 18 hours, serum-deprived for 6 hours, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours ( E and F ). IL-6 mRNA expression was determined by real-time polymerase chain reaction, normalized to 18S rRNA expression, and expressed as fold change with respect to unstimulated control. IL-6 release was determined by ELISA. * P
Figure Legend Snippet: ( A and B ) Semiconfluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours, pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour, and then incubated with medium containing 2.5% fetal bovine serum (FBS) or 2.5% FBS and transforming growth factor (TGF)-β (0.25 ng/ml) for 72 hours ( A ). Alternatively, ASMCs were incubated with adenoviral vectors expressing GFP (Ad-GFP) and wild-type nuclear factor E2-related factor 2 (Ad-Nrf2) (multiplicity of infection 250) for 18 hours, serum-deprived for 6 hours, and then incubated with medium containing 2.5% FBS or 2.5% FBS and TGF-β (0.25 ng/ml) for 72 hours ( B ). DNA synthesis was determined by measuring bromodeoxyuridine (BrdU) incorporation. ( C–F ) Confluent ASMCs were serum-deprived for 24 hours, pretreated with vehicle control or sulforaphane (2–4 μM) for 1 hour, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours ( C and D ). Alternatively, ASMCs were incubated with Ad-GFP or Ad-Nrf2 (MOI 250) for 18 hours, serum-deprived for 6 hours, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours ( E and F ). IL-6 mRNA expression was determined by real-time polymerase chain reaction, normalized to 18S rRNA expression, and expressed as fold change with respect to unstimulated control. IL-6 release was determined by ELISA. * P

Techniques Used: Incubation, Expressing, Infection, DNA Synthesis, BrdU Incorporation Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Airway smooth muscle cells (ASMCs) cultured from bronchoscopic biopsies and transplant airways taken from healthy subjects (n = 5–6) or bronchoscopic biopsies from patients with nonsevere (n = 6) and severe asthma (n = 6–7) were grown to confluence in medium containing 10% fetal bovine serum, and whole-cell protein was extracted. ( A and B ) Nuclear factor E2-related factor 2 (Nrf2) protein expression was determined in whole-cell protein extracts by Western blotting and normalized to β-actin expression. To ensure that all membranes were equally exposed to antibodies, substrate, and X-ray film a control sample (c) was run in each of the gels. ( C ) Nrf2–antioxidant response elements (ARE) binding was determined in whole-cell protein extracts by an ELISA-based TransAM assay. Data were analyzed using Mann-Whitney test.
Figure Legend Snippet: Airway smooth muscle cells (ASMCs) cultured from bronchoscopic biopsies and transplant airways taken from healthy subjects (n = 5–6) or bronchoscopic biopsies from patients with nonsevere (n = 6) and severe asthma (n = 6–7) were grown to confluence in medium containing 10% fetal bovine serum, and whole-cell protein was extracted. ( A and B ) Nuclear factor E2-related factor 2 (Nrf2) protein expression was determined in whole-cell protein extracts by Western blotting and normalized to β-actin expression. To ensure that all membranes were equally exposed to antibodies, substrate, and X-ray film a control sample (c) was run in each of the gels. ( C ) Nrf2–antioxidant response elements (ARE) binding was determined in whole-cell protein extracts by an ELISA-based TransAM assay. Data were analyzed using Mann-Whitney test.

Techniques Used: Cell Culture, Expressing, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

56) Product Images from "Differential chemosensitization of P-glycoprotein overexpressing K562/Adr cells by withaferin A and Siamois polyphenols"

Article Title: Differential chemosensitization of P-glycoprotein overexpressing K562/Adr cells by withaferin A and Siamois polyphenols

Journal: Molecular Cancer

doi: 10.1186/1476-4598-9-99

K562 and K562/Adr cells reveal different nuclear regulation of NFκB, AP1, Nrf2 transcription factors and Sirt1 cofactors . K562 and K562/Adr cells were treated with PMA (0.1 μg/ml) for 30 minutes. Nuclear cell lysates were prepared in SDS-Laemmli sample buffer and extracts were analyzed for protein expression levels by Western analysis of NFκB p65, RelB, cRel, Nrf2, AP1 cjun, junD, Fra1, Sirt1, respectively. Comparable protein loading was verified with Histone H3 antibodies.
Figure Legend Snippet: K562 and K562/Adr cells reveal different nuclear regulation of NFκB, AP1, Nrf2 transcription factors and Sirt1 cofactors . K562 and K562/Adr cells were treated with PMA (0.1 μg/ml) for 30 minutes. Nuclear cell lysates were prepared in SDS-Laemmli sample buffer and extracts were analyzed for protein expression levels by Western analysis of NFκB p65, RelB, cRel, Nrf2, AP1 cjun, junD, Fra1, Sirt1, respectively. Comparable protein loading was verified with Histone H3 antibodies.

Techniques Used: Expressing, Western Blot

K562 and K562/Adr cells show qualitative and quantitative differences in NFκB and AP1 DNA binding profiles . A) K562 and K562/Adr cells were pretreated with PMA (0.1 μg/ml) for 30 minutes. Nuclear lysates were analyzed for NFκB/DNA and AP1/DNA binding with a radiolabeled IL6 κB site- or AP1 motif-containing probe. Binding complexes formed were analyzed by EMSA. Loading of equal amounts of protein was verified by comparison with the binding activity of the repressor molecule recombination signal sequence-binding protein Jκ (RBP-Jκ). Specificity of the various complexes bound is demonstrated by supershift analysis with NFκB- and AP1-specific antibodies, as well as by competition with 100-fold excess cold oligonucleotide. B) K562 and K562/Adr cells were pretreated with 100 μM of quercetin, kaempferol, eriodictyol, WP283, or 6 μM of withaferin A for 2 h followed by incubation with PMA (0.1 μg/ml) for 30 minutes. Cell lysates were fractionated for cytoplasmic and nuclear extracts which were analyzed for NFκB, AP1, or Nrf2-dependent DNA binding with specific radiolabeled probes. Binding complexes formed were analyzed by EMSA.
Figure Legend Snippet: K562 and K562/Adr cells show qualitative and quantitative differences in NFκB and AP1 DNA binding profiles . A) K562 and K562/Adr cells were pretreated with PMA (0.1 μg/ml) for 30 minutes. Nuclear lysates were analyzed for NFκB/DNA and AP1/DNA binding with a radiolabeled IL6 κB site- or AP1 motif-containing probe. Binding complexes formed were analyzed by EMSA. Loading of equal amounts of protein was verified by comparison with the binding activity of the repressor molecule recombination signal sequence-binding protein Jκ (RBP-Jκ). Specificity of the various complexes bound is demonstrated by supershift analysis with NFκB- and AP1-specific antibodies, as well as by competition with 100-fold excess cold oligonucleotide. B) K562 and K562/Adr cells were pretreated with 100 μM of quercetin, kaempferol, eriodictyol, WP283, or 6 μM of withaferin A for 2 h followed by incubation with PMA (0.1 μg/ml) for 30 minutes. Cell lysates were fractionated for cytoplasmic and nuclear extracts which were analyzed for NFκB, AP1, or Nrf2-dependent DNA binding with specific radiolabeled probes. Binding complexes formed were analyzed by EMSA.

Techniques Used: Binding Assay, Activity Assay, Sequencing, Incubation

57) Product Images from "KATP Channel Opener Diazoxide Prevents Neurodegeneration: A New Mechanism of Action via Antioxidative Pathway Activation"

Article Title: KATP Channel Opener Diazoxide Prevents Neurodegeneration: A New Mechanism of Action via Antioxidative Pathway Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0075189

Oral diazoxide administration increases Nrf2 expression and neuronal nuclear translocation in spinal cord of EAE mice. Western blotting of spinal cord total protein showed an increase of Nrf2 immunostain in protein extracts from 0.8/Kg/day oral diazoxide treated EAE mice (either therapeutically and preventively) when compared to vehicle EAE mice (A). Upon quantification, results showed that this increase was significant (B). Double immunofluorescent staining for NeuN (red, C, F) and Nrf2 (green, D, G) of vehicle EAE and 0.8 mg/Kg/day oral diazoxide treated EAE mice showed better NeuN signal preservation (C vs F) and Nrf2 nuclear translocation (E, D vs G, H; white arrows) in the spinal cord ventral horn. n≥6 animal/group. *: p
Figure Legend Snippet: Oral diazoxide administration increases Nrf2 expression and neuronal nuclear translocation in spinal cord of EAE mice. Western blotting of spinal cord total protein showed an increase of Nrf2 immunostain in protein extracts from 0.8/Kg/day oral diazoxide treated EAE mice (either therapeutically and preventively) when compared to vehicle EAE mice (A). Upon quantification, results showed that this increase was significant (B). Double immunofluorescent staining for NeuN (red, C, F) and Nrf2 (green, D, G) of vehicle EAE and 0.8 mg/Kg/day oral diazoxide treated EAE mice showed better NeuN signal preservation (C vs F) and Nrf2 nuclear translocation (E, D vs G, H; white arrows) in the spinal cord ventral horn. n≥6 animal/group. *: p

Techniques Used: Expressing, Translocation Assay, Mouse Assay, Western Blot, Staining, Preserving

Diazoxide increases Nrf2 nuclear translocation in NSC-34 motoneurons and prevents endogenous oxidative damage. Western blot showed an increase of Nrf2 signaling in the nuclear extracts from NSC-34 neurons treated with different doses of diazoxide for 24 h. The higher increase of Nrf2 was found at lower doses (10 and 1 µM) (A). Cell viability of NSC-34 cells was measured after 24 h AAPH oxidative stress activation and results demonstrated that diazoxide treatment effectively ameliorates cell viability at low doses (B). Results expressed as mean ± SEM. n≥4 experiments. *: p
Figure Legend Snippet: Diazoxide increases Nrf2 nuclear translocation in NSC-34 motoneurons and prevents endogenous oxidative damage. Western blot showed an increase of Nrf2 signaling in the nuclear extracts from NSC-34 neurons treated with different doses of diazoxide for 24 h. The higher increase of Nrf2 was found at lower doses (10 and 1 µM) (A). Cell viability of NSC-34 cells was measured after 24 h AAPH oxidative stress activation and results demonstrated that diazoxide treatment effectively ameliorates cell viability at low doses (B). Results expressed as mean ± SEM. n≥4 experiments. *: p

Techniques Used: Translocation Assay, Western Blot, Activation Assay

58) Product Images from "Protective Effects of Curcumin on Renal Oxidative Stress and Lipid Metabolism in a Rat Model of Type 2 Diabetic Nephropathy"

Article Title: Protective Effects of Curcumin on Renal Oxidative Stress and Lipid Metabolism in a Rat Model of Type 2 Diabetic Nephropathy

Journal: Yonsei Medical Journal

doi: 10.3349/ymj.2016.57.3.664

Effect of curcumin on urine SOD and MDA, Nrf2/Keap1, and HO-1 protein expression in the urine or renal cortex. (A) Urinary SOD was significantly increased in the CUR group, compared to the DM group, at 45 weeks of age. (B) Urinary MDA significantly decreased in the CUR group, compared with the DM group. (C) Representative pictures of Nrf2/Keap1 protein ratio and HO-1 expression in renal kidney cortex of 45 weeks of age. (D) Reduced Nrf2/Keap1 protein ratio and HO-1 protein expression related to renal oxidative stress, which were significantly increased in the CUR group. Data expressed as mean±SD. * p
Figure Legend Snippet: Effect of curcumin on urine SOD and MDA, Nrf2/Keap1, and HO-1 protein expression in the urine or renal cortex. (A) Urinary SOD was significantly increased in the CUR group, compared to the DM group, at 45 weeks of age. (B) Urinary MDA significantly decreased in the CUR group, compared with the DM group. (C) Representative pictures of Nrf2/Keap1 protein ratio and HO-1 expression in renal kidney cortex of 45 weeks of age. (D) Reduced Nrf2/Keap1 protein ratio and HO-1 protein expression related to renal oxidative stress, which were significantly increased in the CUR group. Data expressed as mean±SD. * p

Techniques Used: Multiple Displacement Amplification, Expressing

59) Product Images from "Glutathione transferases P1/P2 regulate the timing of signaling pathway activations and cell cycle progression during mouse liver regeneration"

Article Title: Glutathione transferases P1/P2 regulate the timing of signaling pathway activations and cell cycle progression during mouse liver regeneration

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.562

Differential mRNA expression of antioxidant enzymes in the livers of Gstp1/2 +/+ and Gstp1/2 −/− mice after PH. ( a ) mRNA of Nrf2 , MnSOD , catalase , Hmox-1 , Hsp70 , Hif1α , eNOS and iNOS expression in Gstp1/2 +/+ and Gstp1/2 −/− mice at the indicated times after PH. Results are expressed as fold induction compared with the Gstp1/2 +/+ control liver arbitrarily set at 1 and as mean±S.E.M. ( n =3–7 mice/group/time point). * P ≤0.05, ** P ≤0.01, Gstp1/2 −/− versus Gstp1/2 +/+ mice. ( b ) Pool of total proteins from three mice per time point were used for western blotting analysis of eNOS and iNOS in Gstp1/2 +/+ and Gstp1/2 −/− mice at the indicated times after PH. HSC70 is used as a loading control
Figure Legend Snippet: Differential mRNA expression of antioxidant enzymes in the livers of Gstp1/2 +/+ and Gstp1/2 −/− mice after PH. ( a ) mRNA of Nrf2 , MnSOD , catalase , Hmox-1 , Hsp70 , Hif1α , eNOS and iNOS expression in Gstp1/2 +/+ and Gstp1/2 −/− mice at the indicated times after PH. Results are expressed as fold induction compared with the Gstp1/2 +/+ control liver arbitrarily set at 1 and as mean±S.E.M. ( n =3–7 mice/group/time point). * P ≤0.05, ** P ≤0.01, Gstp1/2 −/− versus Gstp1/2 +/+ mice. ( b ) Pool of total proteins from three mice per time point were used for western blotting analysis of eNOS and iNOS in Gstp1/2 +/+ and Gstp1/2 −/− mice at the indicated times after PH. HSC70 is used as a loading control

Techniques Used: Expressing, Mouse Assay, Western Blot

Hepatic localization of NRF2 and MnSOD after PH in Gstp1/2 +/+ and Gstp1/2 −/− mice. Immunolocalization of NRF2 and MnSOD in the Gstp1/2 +/+ and Gstp1/2 −/− livers prior to PH (0 h) and at 24, 36 and 48 h after PH. Bars: 200 μ m
Figure Legend Snippet: Hepatic localization of NRF2 and MnSOD after PH in Gstp1/2 +/+ and Gstp1/2 −/− mice. Immunolocalization of NRF2 and MnSOD in the Gstp1/2 +/+ and Gstp1/2 −/− livers prior to PH (0 h) and at 24, 36 and 48 h after PH. Bars: 200 μ m

Techniques Used: Mouse Assay

60) Product Images from "Metallothionein Is Downstream of Nrf2 and Partially Mediates Sulforaphane Prevention of Diabetic Cardiomyopathy"

Article Title: Metallothionein Is Downstream of Nrf2 and Partially Mediates Sulforaphane Prevention of Diabetic Cardiomyopathy

Journal: Diabetes

doi: 10.2337/db15-1274

SFN-induced Nrf2 downstream gene expression was MT independent. A : The mRNA levels of Nrf2 were detected by qRT-PCR. B : The activation of Nrf2, which was reflected by its nuclear accumulation, was quantified by Western blot of the cardiac nucleus protein. C and D : HO-1 and NQO1 mRNA expression. Data were presented as means ± SD ( n = 6). E : Schematic illustration for SFN protection against type 2 DCM. T2DM induces cardiac oxidative stress, inflammation, and fibrosis, leading from myocardial remodeling and dysfunction to development of DCM. SFN-induced cardiac protection from diabetes via upregulating Nrf2 expression and function, including Nrf2 downstream MT, NQO1, and HO-1 expression. MT, as one of the major Nrf2 downstream targets, plays an important role in the protection by SFN-induced Nrf2 pathway against type 2 DCM. * P
Figure Legend Snippet: SFN-induced Nrf2 downstream gene expression was MT independent. A : The mRNA levels of Nrf2 were detected by qRT-PCR. B : The activation of Nrf2, which was reflected by its nuclear accumulation, was quantified by Western blot of the cardiac nucleus protein. C and D : HO-1 and NQO1 mRNA expression. Data were presented as means ± SD ( n = 6). E : Schematic illustration for SFN protection against type 2 DCM. T2DM induces cardiac oxidative stress, inflammation, and fibrosis, leading from myocardial remodeling and dysfunction to development of DCM. SFN-induced cardiac protection from diabetes via upregulating Nrf2 expression and function, including Nrf2 downstream MT, NQO1, and HO-1 expression. MT, as one of the major Nrf2 downstream targets, plays an important role in the protection by SFN-induced Nrf2 pathway against type 2 DCM. * P

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

Nrf2-KO mice showed increased diabetes-induced cardiac fibrosis and reduced SFN-mediated cardiac protection. A : Cardiac fibrosis, determined by Sirius Red staining of collagen accumulation (collagen is red; scale bar = 50 μm). B : Quantitative analysis of Sirius Red staining for collagen accumulation. C–E : Protein expression of CTGF and TGF-β1 by Western blot. F and G : Cardiac inflammation, measured by mRNA expression of IL-6 and MCP-1 by qRT-PCR. Data were presented as means ± SD ( n = 6). * P
Figure Legend Snippet: Nrf2-KO mice showed increased diabetes-induced cardiac fibrosis and reduced SFN-mediated cardiac protection. A : Cardiac fibrosis, determined by Sirius Red staining of collagen accumulation (collagen is red; scale bar = 50 μm). B : Quantitative analysis of Sirius Red staining for collagen accumulation. C–E : Protein expression of CTGF and TGF-β1 by Western blot. F and G : Cardiac inflammation, measured by mRNA expression of IL-6 and MCP-1 by qRT-PCR. Data were presented as means ± SD ( n = 6). * P

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

SFN completely abolished induced Nrf2 expression and function as well as MT expression. Cardiac oxidative damage, tested by IHC staining for 8-OHdG ( A ; brown considered positive staining; scale bar = 50 μm) followed by a quantitative analysis of the IHC stains ( B ) and lipid peroxidation with MDA assay ( C ). Activation of Nrf2, reflected by its nucleus accumulation (indicated by white arrows) determined by immunofluorescent staining with Nrf2 antibody (red) and nuclear staining with DAPI (blue) on cardiac tissue sections by fluorescence microscope ( D ) (scale bar = 25 μm) and mRNA expression of Nrf2 downstream genes HO-1 and NQO1 ( E ). F : MT expression measured by quantitative PCR and Western blots for its mRNA and protein levels, respectively. Data were presented as means ± SD ( n = 6). * P
Figure Legend Snippet: SFN completely abolished induced Nrf2 expression and function as well as MT expression. Cardiac oxidative damage, tested by IHC staining for 8-OHdG ( A ; brown considered positive staining; scale bar = 50 μm) followed by a quantitative analysis of the IHC stains ( B ) and lipid peroxidation with MDA assay ( C ). Activation of Nrf2, reflected by its nucleus accumulation (indicated by white arrows) determined by immunofluorescent staining with Nrf2 antibody (red) and nuclear staining with DAPI (blue) on cardiac tissue sections by fluorescence microscope ( D ) (scale bar = 25 μm) and mRNA expression of Nrf2 downstream genes HO-1 and NQO1 ( E ). F : MT expression measured by quantitative PCR and Western blots for its mRNA and protein levels, respectively. Data were presented as means ± SD ( n = 6). * P

Techniques Used: Expressing, Immunohistochemistry, Staining, Multiple Displacement Amplification, Activation Assay, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction, Western Blot

SFN-induced Nrf2 expression was MT independent. Cardiac oxidative damage, detected by IHC staining with an anti–8-OHdG antibody ( A ; brown considered positive staining; scale bar = 50 μm) and quantitative analysis of IHC staining ( B ) and lipid peroxidation with MDA assay ( C ). D : Immunofluorescent staining with Nrf2 antibody (red) and nuclear staining with DAPI (blue) on cardiac tissue sections by fluorescence microscope (scale bar = 25 μm). White arrows indicate Nrf2 localized in the nucleus. Data were presented as means ± SD ( n = 6). * P
Figure Legend Snippet: SFN-induced Nrf2 expression was MT independent. Cardiac oxidative damage, detected by IHC staining with an anti–8-OHdG antibody ( A ; brown considered positive staining; scale bar = 50 μm) and quantitative analysis of IHC staining ( B ) and lipid peroxidation with MDA assay ( C ). D : Immunofluorescent staining with Nrf2 antibody (red) and nuclear staining with DAPI (blue) on cardiac tissue sections by fluorescence microscope (scale bar = 25 μm). White arrows indicate Nrf2 localized in the nucleus. Data were presented as means ± SD ( n = 6). * P

Techniques Used: Expressing, Immunohistochemistry, Staining, Multiple Displacement Amplification, Fluorescence, Microscopy

Nrf2-KO mice showed increased diabetes-induced cardiac hypertrophy and reduced SFN-induced cardiac protection. A : Nrf2 mRNA level was detected by qRT-PCR. B : Nuclear Nrf2 protein expression was detected by Western blot. C : Heart size. D and E : Cardiac tissue FITC-conjugated WGA staining and quantification of myocyte cross-sectional areas (scale bar = 25 μm). F and G : qRT-PCR analysis of hypertrophic markers ANP and β-MHC to determine mRNA expression. Data were presented as means ± SD ( n = 6). * P
Figure Legend Snippet: Nrf2-KO mice showed increased diabetes-induced cardiac hypertrophy and reduced SFN-induced cardiac protection. A : Nrf2 mRNA level was detected by qRT-PCR. B : Nuclear Nrf2 protein expression was detected by Western blot. C : Heart size. D and E : Cardiac tissue FITC-conjugated WGA staining and quantification of myocyte cross-sectional areas (scale bar = 25 μm). F and G : qRT-PCR analysis of hypertrophic markers ANP and β-MHC to determine mRNA expression. Data were presented as means ± SD ( n = 6). * P

Techniques Used: Mouse Assay, Quantitative RT-PCR, Expressing, Western Blot, Whole Genome Amplification, Staining, Aqueous Normal-phase Chromatography

61) Product Images from "Zinc Protects Articular Chondrocytes through Changes in Nrf2-Mediated Antioxidants, Cytokines and Matrix Metalloproteinases"

Article Title: Zinc Protects Articular Chondrocytes through Changes in Nrf2-Mediated Antioxidants, Cytokines and Matrix Metalloproteinases

Journal: Nutrients

doi: 10.3390/nu10040471

Effects of MIA and/or zinc on the p-Akt/Nrf2 signaling pathway. ( A ) SW1353 cells were left untreated or treated with 25 μM zinc in the presence or absence of 5 μM MIA, and, after 48 h incubation, the proteins levels of p-Akt and Nrf2 were detected using Western blotting. Actin was used as the internal control. The data in the right panel are expressed as the relative density compared to the untreated cells (control), which was 100%. ( B ) SW1353 cells pretreatment with the 10 μM PI3K inhibitor, LY294002 (LY), for 30 min were treated with 5 μM MIA and/or 25 μM zinc for 48 h and subsequently counted using trypan blue exclusion. The data are expressed as a percentage of the control group. The results are expressed as the means ± S.D. for three separate experiments, each in triplicate. *: p
Figure Legend Snippet: Effects of MIA and/or zinc on the p-Akt/Nrf2 signaling pathway. ( A ) SW1353 cells were left untreated or treated with 25 μM zinc in the presence or absence of 5 μM MIA, and, after 48 h incubation, the proteins levels of p-Akt and Nrf2 were detected using Western blotting. Actin was used as the internal control. The data in the right panel are expressed as the relative density compared to the untreated cells (control), which was 100%. ( B ) SW1353 cells pretreatment with the 10 μM PI3K inhibitor, LY294002 (LY), for 30 min were treated with 5 μM MIA and/or 25 μM zinc for 48 h and subsequently counted using trypan blue exclusion. The data are expressed as a percentage of the control group. The results are expressed as the means ± S.D. for three separate experiments, each in triplicate. *: p

Techniques Used: Incubation, Western Blot

Schematic diagram of zinc effects on MIA-treated chondrocytes. The results of the present study demonstrates that zinc can protect against MIA-increased oxidative stress, pro-inflammatory cytokines (IL-1β), and MMPs through the activation of the Akt/Nrf2 pathway, which upregulates the gene expression of antioxidants, such as Cu/Zn-SOD, Mn-SOD, GPx1, GSH, GCLC and GCLM, and HO-1, leading to the increased antioxidative capacity in defense against MIA-induced oxidative stress. In addition, IL-10 expression is relatively slightly increased by MIA and massively increased after zinc addition, leading to decreased IL-1β and MMPs expression and increased HO-1 expression. Red↑: enhanced by MIA; Red↓: decreased by MIA; Red=: did not change by MIA; Green↑: enhanced by zinc; Green↓: decreased by zinc.
Figure Legend Snippet: Schematic diagram of zinc effects on MIA-treated chondrocytes. The results of the present study demonstrates that zinc can protect against MIA-increased oxidative stress, pro-inflammatory cytokines (IL-1β), and MMPs through the activation of the Akt/Nrf2 pathway, which upregulates the gene expression of antioxidants, such as Cu/Zn-SOD, Mn-SOD, GPx1, GSH, GCLC and GCLM, and HO-1, leading to the increased antioxidative capacity in defense against MIA-induced oxidative stress. In addition, IL-10 expression is relatively slightly increased by MIA and massively increased after zinc addition, leading to decreased IL-1β and MMPs expression and increased HO-1 expression. Red↑: enhanced by MIA; Red↓: decreased by MIA; Red=: did not change by MIA; Green↑: enhanced by zinc; Green↓: decreased by zinc.

Techniques Used: Activation Assay, Expressing

62) Product Images from "Diphlorethohydroxycarmalol Attenuates Methylglyoxal-Induced Oxidative Stress and Advanced Glycation End Product Formation in Human Kidney Cells"

Article Title: Diphlorethohydroxycarmalol Attenuates Methylglyoxal-Induced Oxidative Stress and Advanced Glycation End Product Formation in Human Kidney Cells

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2018/3654095

DPHC activates Nrf2 transcription factor in HEK cells. (a) HEK cells were incubated with the indicated concentrations of DPHC for 24 h. Nuclear and cytoplasmic fractions were prepared, and Western blotting was subsequently performed. (b) HEK cells were transfected with plasmids for EGFP-tagged Nrf2 or EGFP for 33 h and further incubated with or without DPHC (40 μ M) for 24 h. The cells were then fixed and observed by confocal microscopy. Scale bar indicates 10 μ m. (c) HEK cells were incubated with or without DPHC (40 μ M) for 24 h. The cells were stained with an anti-Nrf2 (green) antibody and observed using confocal microscopy. Blue indicates DAPI (nuclear) staining. Scale bar indicates 10 μ m. (d) HEK cells were incubated with or without DPHC (40 μ M) for 1 h and then further incubated with 1 mM MGO for 6 h. The cells were stained with an anti-Nrf2 (green) antibody and observed using confocal microscopy. Blue indicates DAPI (nuclear) staining. Scale bar indicates 10 μ m. ∗ p
Figure Legend Snippet: DPHC activates Nrf2 transcription factor in HEK cells. (a) HEK cells were incubated with the indicated concentrations of DPHC for 24 h. Nuclear and cytoplasmic fractions were prepared, and Western blotting was subsequently performed. (b) HEK cells were transfected with plasmids for EGFP-tagged Nrf2 or EGFP for 33 h and further incubated with or without DPHC (40 μ M) for 24 h. The cells were then fixed and observed by confocal microscopy. Scale bar indicates 10 μ m. (c) HEK cells were incubated with or without DPHC (40 μ M) for 24 h. The cells were stained with an anti-Nrf2 (green) antibody and observed using confocal microscopy. Blue indicates DAPI (nuclear) staining. Scale bar indicates 10 μ m. (d) HEK cells were incubated with or without DPHC (40 μ M) for 1 h and then further incubated with 1 mM MGO for 6 h. The cells were stained with an anti-Nrf2 (green) antibody and observed using confocal microscopy. Blue indicates DAPI (nuclear) staining. Scale bar indicates 10 μ m. ∗ p

Techniques Used: Incubation, Western Blot, Transfection, Confocal Microscopy, Staining

DPHC did not affect antioxidant, detoxifying, ROS, AGEs, or protein carbonyl content in Nrf2-knockdown HEK cells. HEK cells were transfected with Nrf2 siRNA for 36 h. The transfected cells were incubated with 40 μ M DPHC for 24 h. (a) qRT-PCR and Western blot were performed for nuclear factor (erythroid-derived 2)-like 2 (Nrf2) mRNA and protein expression. (b) qRT-PCR was performed for superoxide dismutase (SOD) 1 and 2, catalase (CAT), heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase (NQO)1, glutamylcysteine synthetase (GCL)c, and GCLm. (c–g) HEK cells were transfected with Nrf2 siRNA for 36 h. The transfected cells were incubated with or without 40 μ M DPHC for 1 h and then further incubated with or without 1 mM MGO for 24 h. (c) ROS production was measured by flow cytometry. (d) AGE content was measured. (e) Protein carbonyl content was measured. (f) Nrf2 mRNA expression was measured by qRT-PCR. (g) Glo-1 (glyoxalase 1) mRNA expression was measured by qRT-PCR. Experiments were performed in triplicate. ∗ p
Figure Legend Snippet: DPHC did not affect antioxidant, detoxifying, ROS, AGEs, or protein carbonyl content in Nrf2-knockdown HEK cells. HEK cells were transfected with Nrf2 siRNA for 36 h. The transfected cells were incubated with 40 μ M DPHC for 24 h. (a) qRT-PCR and Western blot were performed for nuclear factor (erythroid-derived 2)-like 2 (Nrf2) mRNA and protein expression. (b) qRT-PCR was performed for superoxide dismutase (SOD) 1 and 2, catalase (CAT), heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase (NQO)1, glutamylcysteine synthetase (GCL)c, and GCLm. (c–g) HEK cells were transfected with Nrf2 siRNA for 36 h. The transfected cells were incubated with or without 40 μ M DPHC for 1 h and then further incubated with or without 1 mM MGO for 24 h. (c) ROS production was measured by flow cytometry. (d) AGE content was measured. (e) Protein carbonyl content was measured. (f) Nrf2 mRNA expression was measured by qRT-PCR. (g) Glo-1 (glyoxalase 1) mRNA expression was measured by qRT-PCR. Experiments were performed in triplicate. ∗ p

Techniques Used: Transfection, Incubation, Quantitative RT-PCR, Western Blot, Derivative Assay, Expressing, Flow Cytometry, Cytometry

Possible mechanisms for the inhibition of AGE accumulation by DPHC in MGO-treated HEK cells. DPHC promotes the expression of not only antioxidant and phase II detoxifying enzymes but also Glo-1 by mediating Nrf2 activation. This inhibits oxidative stress and formation of AGEs, effectively attenuating cell damage. DPHC: diphlorethohydroxycarmalol; MGO: methylglyoxal; Nrf2: nuclear factor erythroid 2-related factor 2; ARE: antioxidant response element; ROS: reactive oxygen species; AGEs: advanced glycation end products; Glo-1: glyoxalase 1; SOD: superoxide dismutase; CAT: catalase; HO-1: heme oxygenase-1; NQO: NAD(P)H quinone dehydrogenase 1; GCL: γ -glutamylcysteine synthetases.
Figure Legend Snippet: Possible mechanisms for the inhibition of AGE accumulation by DPHC in MGO-treated HEK cells. DPHC promotes the expression of not only antioxidant and phase II detoxifying enzymes but also Glo-1 by mediating Nrf2 activation. This inhibits oxidative stress and formation of AGEs, effectively attenuating cell damage. DPHC: diphlorethohydroxycarmalol; MGO: methylglyoxal; Nrf2: nuclear factor erythroid 2-related factor 2; ARE: antioxidant response element; ROS: reactive oxygen species; AGEs: advanced glycation end products; Glo-1: glyoxalase 1; SOD: superoxide dismutase; CAT: catalase; HO-1: heme oxygenase-1; NQO: NAD(P)H quinone dehydrogenase 1; GCL: γ -glutamylcysteine synthetases.

Techniques Used: Inhibition, Expressing, Activation Assay

63) Product Images from "Evidence for a novel antioxidant function and isoform-specific regulation of the human p66Shc gene"

Article Title: Evidence for a novel antioxidant function and isoform-specific regulation of the human p66Shc gene

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E13-11-0666

p66Shc deficiency impairs expression of other ARE-dependent antioxidant genes. (A) si Control– or p66Shc siRNA-1–transfected K562 cells were treated with 20 μM hemin for 1, 2, and 3 d. Whole-cell lysates were used in Western blots for Shc, ferritin H (FH), ferritin L (FL), NQO1, GSTπ, thioredoxin-1 (Trx), β-actin, and GAPDH, and nuclear extracts were used for Nrf2 and lamin B1. (B) siRNA-transfected K562 cells were treated with 20 μM hemin for 36 h, and real-time PCR was performed for ferritin H, NQO1, and Trx mRNA. Data are presented as relative mRNA expression (si Control/day 0 is defined as 1.0) after normalization with RPL13A (ribosomal protein L13A). Data are shown as mean ± SD ( n = 3). (C) Shc and GAPDH Western blots and Nrf2 and lamin B1 Western blots were performed using whole-cell lysates (Total) and nuclear extracts (Nuc.) of siRNA-transfected K562 cells treated with 20 μM hemin for 36 h, respectively. Nuclear extracts were also used for pull-down assay. Nrf2 binding to biotinylated double-strand ferritin H ARE (−4067 to −4046 base pairs) probe was detected by Western blot using an Nrf2 antibody. Coomassie brilliant blue (CBB) staining is shown for verification of equal loading. (D) siRNA-transfected K562 cells treated with 20 μM hemin for 36 h were subjected to ChIP assays with control IgG or Nrf2 antibody, followed by real-time PCR using ferritin L and NQO1 primers spanning the ARE region. Data were normalized by input DNA and are shown as mean ± SD ( n = 3).
Figure Legend Snippet: p66Shc deficiency impairs expression of other ARE-dependent antioxidant genes. (A) si Control– or p66Shc siRNA-1–transfected K562 cells were treated with 20 μM hemin for 1, 2, and 3 d. Whole-cell lysates were used in Western blots for Shc, ferritin H (FH), ferritin L (FL), NQO1, GSTπ, thioredoxin-1 (Trx), β-actin, and GAPDH, and nuclear extracts were used for Nrf2 and lamin B1. (B) siRNA-transfected K562 cells were treated with 20 μM hemin for 36 h, and real-time PCR was performed for ferritin H, NQO1, and Trx mRNA. Data are presented as relative mRNA expression (si Control/day 0 is defined as 1.0) after normalization with RPL13A (ribosomal protein L13A). Data are shown as mean ± SD ( n = 3). (C) Shc and GAPDH Western blots and Nrf2 and lamin B1 Western blots were performed using whole-cell lysates (Total) and nuclear extracts (Nuc.) of siRNA-transfected K562 cells treated with 20 μM hemin for 36 h, respectively. Nuclear extracts were also used for pull-down assay. Nrf2 binding to biotinylated double-strand ferritin H ARE (−4067 to −4046 base pairs) probe was detected by Western blot using an Nrf2 antibody. Coomassie brilliant blue (CBB) staining is shown for verification of equal loading. (D) siRNA-transfected K562 cells treated with 20 μM hemin for 36 h were subjected to ChIP assays with control IgG or Nrf2 antibody, followed by real-time PCR using ferritin L and NQO1 primers spanning the ARE region. Data were normalized by input DNA and are shown as mean ± SD ( n = 3).

Techniques Used: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Pull Down Assay, Binding Assay, Staining, Chromatin Immunoprecipitation

Nrf2 knockdown blocks hemin-induced transcriptional activation of the p66Shc gene. (A) K562 cells were treated with 0 and 40 μM hemin for 24 h after Nrf2 knockdown using Nrf2-targeted siRNA-1 or -2 (si Nrf2-1 or si Nrf2-2). Real-time PCR was performed for p66Shc and Nrf2 mRNA expression. Results are presented as relative mRNA expression (nontargeted control siRNA [si Control], no hemin as 1.0) and normalized by GAPDH. p values were calculated using Student's t test. Asterisk indicates significant difference; * p = 0.01, ** p = 0.045. Data are shown as mean ± SD ( n > 4). (B) K562 cells transfected with a si Control (si Ctrl) or si Nrf2-1 were treated with 0, 10, 20, or 40 μM hemin for 24 h. Top, Western blots using anti-Shc and anti-GAPDH antibodies. Bottom, Nrf2 mRNA levels measured by real-time PCR after normalization with GAPDH mRNA. Data are shown as mean ± SD ( n > 4). (C) K562 cells were transfected with pCMV or pCMV-Nrf2 plasmid and treated with 0, 20, and 40 μM hemin for 24 h. Whole-cell lysate (Total) and nuclear fraction (Nuc.) were used for Shc and GAPDH Western blot and Nrf2 and lamin B1 Western blot, respectively. (D) K562 cells were treated with 0 and 50 μM hemin for 12 h after Nrf2 knockdown, and ChIP assays were performed with control IgG or an anti–RNA polymerase II antibody (RNAPII), followed by real-time PCR using primers spanning the p66Shc transcriptional start site. Data were normalized by input DNA and are shown as mean ± SD ( n = 3). p values were calculated using Student's t test. Asterisk indicates significant difference; * p = 0.002, ** p = 0.005.
Figure Legend Snippet: Nrf2 knockdown blocks hemin-induced transcriptional activation of the p66Shc gene. (A) K562 cells were treated with 0 and 40 μM hemin for 24 h after Nrf2 knockdown using Nrf2-targeted siRNA-1 or -2 (si Nrf2-1 or si Nrf2-2). Real-time PCR was performed for p66Shc and Nrf2 mRNA expression. Results are presented as relative mRNA expression (nontargeted control siRNA [si Control], no hemin as 1.0) and normalized by GAPDH. p values were calculated using Student's t test. Asterisk indicates significant difference; * p = 0.01, ** p = 0.045. Data are shown as mean ± SD ( n > 4). (B) K562 cells transfected with a si Control (si Ctrl) or si Nrf2-1 were treated with 0, 10, 20, or 40 μM hemin for 24 h. Top, Western blots using anti-Shc and anti-GAPDH antibodies. Bottom, Nrf2 mRNA levels measured by real-time PCR after normalization with GAPDH mRNA. Data are shown as mean ± SD ( n > 4). (C) K562 cells were transfected with pCMV or pCMV-Nrf2 plasmid and treated with 0, 20, and 40 μM hemin for 24 h. Whole-cell lysate (Total) and nuclear fraction (Nuc.) were used for Shc and GAPDH Western blot and Nrf2 and lamin B1 Western blot, respectively. (D) K562 cells were treated with 0 and 50 μM hemin for 12 h after Nrf2 knockdown, and ChIP assays were performed with control IgG or an anti–RNA polymerase II antibody (RNAPII), followed by real-time PCR using primers spanning the p66Shc transcriptional start site. Data were normalized by input DNA and are shown as mean ± SD ( n = 3). p values were calculated using Student's t test. Asterisk indicates significant difference; * p = 0.002, ** p = 0.005.

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

Graphic summary. Transcription of the human p66Shc, but not p52Shc or p46Shc, is regulated by Nrf2. Phosphorylation of p66Shc at Ser-36, known as an active mitochondrial form, was induced in K562 cells treated with hemin but still localized in the cytoplasm. p66Shc serves as an antioxidant protein by maintaining up-regulation of other ARE-dependent genes (NQO1, Trx, ferritin H and L, γ-globin) and is also involved in erythroid differentiation. The present work demonstrates the isoform-specific regulation of the Shc gene by the Nrf2-ARE system and a new antioxidant function of cytoplasmic p66Shc involved in cellular oxidative stress response and differentiation.
Figure Legend Snippet: Graphic summary. Transcription of the human p66Shc, but not p52Shc or p46Shc, is regulated by Nrf2. Phosphorylation of p66Shc at Ser-36, known as an active mitochondrial form, was induced in K562 cells treated with hemin but still localized in the cytoplasm. p66Shc serves as an antioxidant protein by maintaining up-regulation of other ARE-dependent genes (NQO1, Trx, ferritin H and L, γ-globin) and is also involved in erythroid differentiation. The present work demonstrates the isoform-specific regulation of the Shc gene by the Nrf2-ARE system and a new antioxidant function of cytoplasmic p66Shc involved in cellular oxidative stress response and differentiation.

Techniques Used:

Nrf2 binds the human p66Shc promoter containing the ARE and AP1-like sequences. (A) K562 cells were treated with 20 μM hemin for 36 h, and nuclear (Nuc.) and cytoplasmic (Cyto.) fractions were used for Western blot with Nrf2, lamin B1, or LDH antibody. Lamin B1 and LDH are nuclear and cytoplasmic fraction markers, respectively. (B) Schematic of the 5′-promoter region of the human p66Shc gene, which contains an ARE and an AP1-like sequence. n = any base; M = A or C; R = A or G; Y = C or T. Asterisks indicate matched bases. (C) K562 cells treated with 0, 20, 40, and 75 μM hemin for 12 h were subjected to ChIP assays with control IgG or Nrf2 antibody, followed by real-time PCR using p66Shc primers spanning the ARE, AP1-like, a 2.3-kb upstream 5′-promoter region, and a β-globin promoter region. Data were normalized by input DNA and are shown as mean ± SD ( n = 3). (D) Nuclear extracts of K562 cells treated with 20 μM hemin for 36 h were used for pull-down assay. Nrf2 binding to a biotinylated double-strand p66Shc ARE (−107 to −82 base pairs) or a p66Shc AP1-like (−291 to −266 base pairs) probe was detected by Western blot using an Nrf2 antibody. A human ferritin H ARE (–4433 to –4412 base pairs) probe was used as a positive control and no DNA probe in the binding reaction as a negative control. Coomassie brilliant blue (CBB) staining is shown for verification of equal loading. (E) K562 cells were transfected with wild-type or mutant pGL3 p66Shc-450/+60 plasmid and incubated with 0, 20, and 40 μM hemin for 24 h. Firefly luciferase activity was normalized by Renilla luciferase activity and presented as relative to nontreated wild-type p66Shc-450/+60 plasmid. Top, asterisks indicate mutated nucleic acid. Bottom chart, asterisks indicate significantly different from nontreated cells transfected with wild-type p66Shc-450/+60 plasmid. * p
Figure Legend Snippet: Nrf2 binds the human p66Shc promoter containing the ARE and AP1-like sequences. (A) K562 cells were treated with 20 μM hemin for 36 h, and nuclear (Nuc.) and cytoplasmic (Cyto.) fractions were used for Western blot with Nrf2, lamin B1, or LDH antibody. Lamin B1 and LDH are nuclear and cytoplasmic fraction markers, respectively. (B) Schematic of the 5′-promoter region of the human p66Shc gene, which contains an ARE and an AP1-like sequence. n = any base; M = A or C; R = A or G; Y = C or T. Asterisks indicate matched bases. (C) K562 cells treated with 0, 20, 40, and 75 μM hemin for 12 h were subjected to ChIP assays with control IgG or Nrf2 antibody, followed by real-time PCR using p66Shc primers spanning the ARE, AP1-like, a 2.3-kb upstream 5′-promoter region, and a β-globin promoter region. Data were normalized by input DNA and are shown as mean ± SD ( n = 3). (D) Nuclear extracts of K562 cells treated with 20 μM hemin for 36 h were used for pull-down assay. Nrf2 binding to a biotinylated double-strand p66Shc ARE (−107 to −82 base pairs) or a p66Shc AP1-like (−291 to −266 base pairs) probe was detected by Western blot using an Nrf2 antibody. A human ferritin H ARE (–4433 to –4412 base pairs) probe was used as a positive control and no DNA probe in the binding reaction as a negative control. Coomassie brilliant blue (CBB) staining is shown for verification of equal loading. (E) K562 cells were transfected with wild-type or mutant pGL3 p66Shc-450/+60 plasmid and incubated with 0, 20, and 40 μM hemin for 24 h. Firefly luciferase activity was normalized by Renilla luciferase activity and presented as relative to nontreated wild-type p66Shc-450/+60 plasmid. Top, asterisks indicate mutated nucleic acid. Bottom chart, asterisks indicate significantly different from nontreated cells transfected with wild-type p66Shc-450/+60 plasmid. * p

Techniques Used: Western Blot, Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Pull Down Assay, Binding Assay, Positive Control, Negative Control, Staining, Transfection, Mutagenesis, Plasmid Preparation, Incubation, Luciferase, Activity Assay

64) Product Images from "Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway"

Article Title: Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S110505

UA sensitizes HepG2/DDP cells to low-dose cisplatin via inhibition of Nrf2/ARE signaling pathway. Notes: ( A ) HepG2/DDP cells were transfected with Nrf2 siRNA (si-Nrf2) or negative control (si-Con), or ( C ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with 8.92 μg/mL cisplatin (IC 30 of cisplatin for HepG2/DDP cells) and/or UA (2.25 μg/mL) for 48 hours. The level of Nrf2, HO-1, NQO1, and GST was detected by Western blot analysis. ( B ) HepG2/DDP cells were transfected with si-Nrf2 or si-Con, or ( D ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with series concentration of cisplatin (2–512 μg/mL) and/or UA (2.25 μg/mL) for 48 hours. The inhibition rate of cell was detected by CCK8 assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: ARE, antioxidant response element; CCK8, Cell Counting Kit 8; cDNA, complementary DNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin–resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; IC 30 , 30% inhibitory concentration; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; siRNA, small interfering RNA; UA, ursolic acid.
Figure Legend Snippet: UA sensitizes HepG2/DDP cells to low-dose cisplatin via inhibition of Nrf2/ARE signaling pathway. Notes: ( A ) HepG2/DDP cells were transfected with Nrf2 siRNA (si-Nrf2) or negative control (si-Con), or ( C ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with 8.92 μg/mL cisplatin (IC 30 of cisplatin for HepG2/DDP cells) and/or UA (2.25 μg/mL) for 48 hours. The level of Nrf2, HO-1, NQO1, and GST was detected by Western blot analysis. ( B ) HepG2/DDP cells were transfected with si-Nrf2 or si-Con, or ( D ) HepG2/DDP cells were transfected with Nrf2 cDNA or empty vector (Vector), then treated with series concentration of cisplatin (2–512 μg/mL) and/or UA (2.25 μg/mL) for 48 hours. The inhibition rate of cell was detected by CCK8 assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: ARE, antioxidant response element; CCK8, Cell Counting Kit 8; cDNA, complementary DNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin–resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; IC 30 , 30% inhibitory concentration; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; siRNA, small interfering RNA; UA, ursolic acid.

Techniques Used: Inhibition, Transfection, Negative Control, Plasmid Preparation, Western Blot, Concentration Assay, CCK-8 Assay, Cell Counting, Standard Deviation, Small Interfering RNA

Nrf2 was overexpressed in cisplatin-resistant human hepatocellular carcinoma HepG2/DDP cells. Notes: ( A ) HepG2 cells were treated with series concentration of cisplatin (0.1–25.6 μg/mL) for 48 hours. ( B ) HepG2/DDP cells were treated with series concentration of cisplatin (2–512 μg/mL) for 48 hours. ( C ) The level of Nrf2 and its downstream target genes HO-1, NQO1, and GST in HepG2 and HepG2/DDP cells was detected by Western blot assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2, hepatocellular carcinoma cell line; HepG2/DDP, cisplatin-resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation.
Figure Legend Snippet: Nrf2 was overexpressed in cisplatin-resistant human hepatocellular carcinoma HepG2/DDP cells. Notes: ( A ) HepG2 cells were treated with series concentration of cisplatin (0.1–25.6 μg/mL) for 48 hours. ( B ) HepG2/DDP cells were treated with series concentration of cisplatin (2–512 μg/mL) for 48 hours. ( C ) The level of Nrf2 and its downstream target genes HO-1, NQO1, and GST in HepG2 and HepG2/DDP cells was detected by Western blot assay. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2, hepatocellular carcinoma cell line; HepG2/DDP, cisplatin-resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation.

Techniques Used: Concentration Assay, Western Blot, Standard Deviation

UA–cisplatin combination downregulates Nrf2 and its substrates. Notes: The protein expression levels of Nrf2, HO-1, NQO1, and GST of HepG2/DDP cells treated with 8.92 μg/mL cisplatin and/or UA (2.25 μg/mL) for 48 hours were detected by Western blot analysis. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin-resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; UA, ursolic acid.
Figure Legend Snippet: UA–cisplatin combination downregulates Nrf2 and its substrates. Notes: The protein expression levels of Nrf2, HO-1, NQO1, and GST of HepG2/DDP cells treated with 8.92 μg/mL cisplatin and/or UA (2.25 μg/mL) for 48 hours were detected by Western blot analysis. Results are representative of three different experiments, and they are expressed as mean ± SD. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S -transferase; HepG2/DDP, cisplatin-resistant hepatocellular carcinoma cell line; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid-2-related factor 2; SD, standard deviation; UA, ursolic acid.

Techniques Used: Expressing, Western Blot, Standard Deviation

65) Product Images from "DNA Protecting Activities of Nymphaea nouchali (Burm. f) Flower Extract Attenuate t-BHP-Induced Oxidative Stress Cell Death through Nrf2-Mediated Induction of Heme Oxygenase-1 Expression by Activating MAP-Kinases"

Article Title: DNA Protecting Activities of Nymphaea nouchali (Burm. f) Flower Extract Attenuate t-BHP-Induced Oxidative Stress Cell Death through Nrf2-Mediated Induction of Heme Oxygenase-1 Expression by Activating MAP-Kinases

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18102069

Effect of NNFE on nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated HO-1 expression. RAW 264.7 cells were pretreated for 24 h with NNFE. The mRNA expression ( A ) and protein expression ( B ) of Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) were measured by RT-PCR and western blot, respectively. ( C ) The time-dependent effect on the protein levels of cytosolic and nuclear Nrf2 was analyzed by western blot. ( D ) Cells were treated with an Nrf2 inhibitor (brusatol) with and without NNFE, and Nrf2 and HO-1 protein levels were analyzed by western blot. ( E ) Cells were treated with a siRNA of Nrf2 (si-Nrf2) with and without NNFE, and Nrf2 and HO-1 protein levels were analyzed by western blot. Statistical values are expressed as the mean ± SD ( n = 3). # p
Figure Legend Snippet: Effect of NNFE on nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated HO-1 expression. RAW 264.7 cells were pretreated for 24 h with NNFE. The mRNA expression ( A ) and protein expression ( B ) of Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) were measured by RT-PCR and western blot, respectively. ( C ) The time-dependent effect on the protein levels of cytosolic and nuclear Nrf2 was analyzed by western blot. ( D ) Cells were treated with an Nrf2 inhibitor (brusatol) with and without NNFE, and Nrf2 and HO-1 protein levels were analyzed by western blot. ( E ) Cells were treated with a siRNA of Nrf2 (si-Nrf2) with and without NNFE, and Nrf2 and HO-1 protein levels were analyzed by western blot. Statistical values are expressed as the mean ± SD ( n = 3). # p

Techniques Used: Translocation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

NNFE extract activates the translocation of Nrf2 by activating extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38. ( A ) RAW 264.7 cells were pretreated with 10 μg/mL NNFE for the indicated time and kinase activation was analyzed by western blot. ( B ) Cells were treated with 10 μg/mL NNFE in the presence and absence of specific inhibitor U0126 and SB239063 and the protein level of Nrf2 and HO-1 were analyzed by western blot. Following co-treatment with NNFE, U0126, and SB239063 for 12 h, cells were challenged with t -BHP for an additional 12 h, and then cell viability ( C ) and intracellular ROS generation ( D ) were analyzed. Statistical values are expressed as the mean ± SD ( n = 3). # p
Figure Legend Snippet: NNFE extract activates the translocation of Nrf2 by activating extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38. ( A ) RAW 264.7 cells were pretreated with 10 μg/mL NNFE for the indicated time and kinase activation was analyzed by western blot. ( B ) Cells were treated with 10 μg/mL NNFE in the presence and absence of specific inhibitor U0126 and SB239063 and the protein level of Nrf2 and HO-1 were analyzed by western blot. Following co-treatment with NNFE, U0126, and SB239063 for 12 h, cells were challenged with t -BHP for an additional 12 h, and then cell viability ( C ) and intracellular ROS generation ( D ) were analyzed. Statistical values are expressed as the mean ± SD ( n = 3). # p

Techniques Used: Translocation Assay, Activation Assay, Western Blot

66) Product Images from "Effects of dimethyl fumarate on neuroprotection and immunomodulation"

Article Title: Effects of dimethyl fumarate on neuroprotection and immunomodulation

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-9-163

Dimethyl fumarate (DMF) protects from oxidative stress by enhancing nuclear factor erythroid 2-related factor 2 (Nrf2) abundance and translocation to the nucleus in a time- and concentration-dependent manner. A ) DMF protects from oxidative glutamate toxicity, a model of endogenous oxidative stress where extracellular glutamate blocks the cystine import ultimately causing glutathione (GSH) depletion and cell death. Primary cortical cultures or hippocampal HT22 cells were preincubated with 10 μM DMF, monomethylfumarate (MMF) or vehicle for 24 h and exposed to the indicated concentrations of glutamate for 24 h before cell viability was measured by the Cell Titer Blue (CTB) assay. B ) DMF and MMF increase cellular GSH concentrations. HT22 cells were treated with 10 μM DMF, MMF or vehicle for 24 h and exposed to the indicated concentrations of glutamate for 8 h before intracellular glutathione was measured enzymatically. C - F ) Dose and time-course of DMF and MMF effect on oxidative glutamate toxicity. HT22 cells were treated for the indicated times and concentrations with DMF, MMF or vehicle before addition of glutamate. Viability was quantitated 24 h later as described above. G ) Time course of DMF effects on glutathione content. HT22 cells were incubated with 10 μM DMF for the indicated periods of time before intracellular glutathione was measured enzymatically. H ) DMF enhances Nrf2 abundance quantitated by immunoblots done on nuclear fractions from HT22 cells treated with the indicated concentrations of DMF for 4 h. I ) and J ) DMF induces nuclear localization of Nrf2 but has no effect on the nuclear translocation of NF-κB as shown by high content imaging. I ) HT22 cells were treated with vehicle (n = 9,561 cells), 10 μM DMF for 24 h (n = 8,170 cells) or with 25 μM TBHQ (n = 3,281 cells) as positive control for 4 h. J ) HT22 cells were treated with vehicle or 10 ng/ml TNFα in the presence or absence of 10 μM DMF (vehicle n = 1,048 cells, DMF n = 943 cells, TNFα n = 1,410 cells, DMF + TNFα n = 1,085 cells). Cells were fixed, stained and nuclear localization analyzed by immunocytochemistry. K ) DMF has no effect on GSH levels in fibroblasts derived from Nrf2-deficient mice. Cells were treated with 10 μM DMF (black bars) or vehicle (white bars) for 24 h before GSH was measured enzymatically. Graphs of all experiments represent the means ± standard error of the mean (SEM) of three independent experiments performed in triplicate. * P
Figure Legend Snippet: Dimethyl fumarate (DMF) protects from oxidative stress by enhancing nuclear factor erythroid 2-related factor 2 (Nrf2) abundance and translocation to the nucleus in a time- and concentration-dependent manner. A ) DMF protects from oxidative glutamate toxicity, a model of endogenous oxidative stress where extracellular glutamate blocks the cystine import ultimately causing glutathione (GSH) depletion and cell death. Primary cortical cultures or hippocampal HT22 cells were preincubated with 10 μM DMF, monomethylfumarate (MMF) or vehicle for 24 h and exposed to the indicated concentrations of glutamate for 24 h before cell viability was measured by the Cell Titer Blue (CTB) assay. B ) DMF and MMF increase cellular GSH concentrations. HT22 cells were treated with 10 μM DMF, MMF or vehicle for 24 h and exposed to the indicated concentrations of glutamate for 8 h before intracellular glutathione was measured enzymatically. C - F ) Dose and time-course of DMF and MMF effect on oxidative glutamate toxicity. HT22 cells were treated for the indicated times and concentrations with DMF, MMF or vehicle before addition of glutamate. Viability was quantitated 24 h later as described above. G ) Time course of DMF effects on glutathione content. HT22 cells were incubated with 10 μM DMF for the indicated periods of time before intracellular glutathione was measured enzymatically. H ) DMF enhances Nrf2 abundance quantitated by immunoblots done on nuclear fractions from HT22 cells treated with the indicated concentrations of DMF for 4 h. I ) and J ) DMF induces nuclear localization of Nrf2 but has no effect on the nuclear translocation of NF-κB as shown by high content imaging. I ) HT22 cells were treated with vehicle (n = 9,561 cells), 10 μM DMF for 24 h (n = 8,170 cells) or with 25 μM TBHQ (n = 3,281 cells) as positive control for 4 h. J ) HT22 cells were treated with vehicle or 10 ng/ml TNFα in the presence or absence of 10 μM DMF (vehicle n = 1,048 cells, DMF n = 943 cells, TNFα n = 1,410 cells, DMF + TNFα n = 1,085 cells). Cells were fixed, stained and nuclear localization analyzed by immunocytochemistry. K ) DMF has no effect on GSH levels in fibroblasts derived from Nrf2-deficient mice. Cells were treated with 10 μM DMF (black bars) or vehicle (white bars) for 24 h before GSH was measured enzymatically. Graphs of all experiments represent the means ± standard error of the mean (SEM) of three independent experiments performed in triplicate. * P

Techniques Used: Translocation Assay, Concentration Assay, CtB Assay, Incubation, Western Blot, Imaging, Positive Control, Staining, Immunocytochemistry, Derivative Assay, Mouse Assay

67) Product Images from "Targeting of Nrf2 induces DNA damage signaling and protects colonic epithelial cells from ionizing radiation"

Article Title: Targeting of Nrf2 induces DNA damage signaling and protects colonic epithelial cells from ionizing radiation

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1207718109

CDDO-EA increases mouse survival after an acute lethal dose of TBI. ( A ) CDDO-EA stabilizes and activates Nrf2 in mouse colon tissues. A CDDO-EA diet was provided to unirradiated WT mice for 1, 2, or 3 d, and the colon tissues were then lysed. Total Nrf2
Figure Legend Snippet: CDDO-EA increases mouse survival after an acute lethal dose of TBI. ( A ) CDDO-EA stabilizes and activates Nrf2 in mouse colon tissues. A CDDO-EA diet was provided to unirradiated WT mice for 1, 2, or 3 d, and the colon tissues were then lysed. Total Nrf2

Techniques Used: Mouse Assay

Pretreatment with a synthetic triterpenoid before IR activates Nrf2, which regulates transcription of the antioxidant enzyme HO-1 and the DNA repair protein, p53 binding protein-1 (53BP1). Increased levels of HO-1 and 53BP1 protect colonic epithelial
Figure Legend Snippet: Pretreatment with a synthetic triterpenoid before IR activates Nrf2, which regulates transcription of the antioxidant enzyme HO-1 and the DNA repair protein, p53 binding protein-1 (53BP1). Increased levels of HO-1 and 53BP1 protect colonic epithelial

Techniques Used: Binding Assay

BARD protects HCECs from irradiation through Nrf2. ( A ) Clonogenic survival for countermeasure effect. HCECs were treated with BARD for 18 h, and cells were then irradiated with the indicated radiation doses. ( B ) Establishment of Nrf2 knockdown cell lines.
Figure Legend Snippet: BARD protects HCECs from irradiation through Nrf2. ( A ) Clonogenic survival for countermeasure effect. HCECs were treated with BARD for 18 h, and cells were then irradiated with the indicated radiation doses. ( B ) Establishment of Nrf2 knockdown cell lines.

Techniques Used: Irradiation

Nrf2 increases 53BP1 expression by BARD treatment. ChIP-qPCR analysis of Nrf2 occupancy on the 53BP1 ( A ) and HO-1 ( B ) promoters using unirradiated cells following treatment with vehicle or BARD for 18 h. Data shown are from two separate triplicate experiments.
Figure Legend Snippet: Nrf2 increases 53BP1 expression by BARD treatment. ChIP-qPCR analysis of Nrf2 occupancy on the 53BP1 ( A ) and HO-1 ( B ) promoters using unirradiated cells following treatment with vehicle or BARD for 18 h. Data shown are from two separate triplicate experiments.

Techniques Used: Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

68) Product Images from "Apigenin Reactivates Nrf2 Anti-oxidative Stress Signaling in Mouse Skin Epidermal JB6 P + Cells Through Epigenetics Modifications"

Article Title: Apigenin Reactivates Nrf2 Anti-oxidative Stress Signaling in Mouse Skin Epidermal JB6 P + Cells Through Epigenetics Modifications

Journal: The AAPS Journal

doi: 10.1208/s12248-014-9613-8

API increases the level of the nuclear translocation of the Nrf2 protein. JB6 P + cells were treated with DMSO and API for 5 days. Asterisk and number sign indicate significant differences ( p
Figure Legend Snippet: API increases the level of the nuclear translocation of the Nrf2 protein. JB6 P + cells were treated with DMSO and API for 5 days. Asterisk and number sign indicate significant differences ( p

Techniques Used: Translocation Assay

API decreases the methylation level of 15 CpG sites in the Nrf2 promoter in JB6 P + cells. The methylation level at the 15 CpG sites in the Nrf2 promoter was determined using bisulfite genomic sequencing (BGS), as described in the “
Figure Legend Snippet: API decreases the methylation level of 15 CpG sites in the Nrf2 promoter in JB6 P + cells. The methylation level at the 15 CpG sites in the Nrf2 promoter was determined using bisulfite genomic sequencing (BGS), as described in the “

Techniques Used: Methylation, Genomic Sequencing

API increases the level of mRNA and protein expression of Nrf2 and the Nrf2 downstream gene, NQO1. a Relative fold changes of mRNA expression of Nrf2 and NQO1 in JB6 P + cells treated with API, DMSO, and the DNMT/HDAC inhibitor (5-aza/TSA)
Figure Legend Snippet: API increases the level of mRNA and protein expression of Nrf2 and the Nrf2 downstream gene, NQO1. a Relative fold changes of mRNA expression of Nrf2 and NQO1 in JB6 P + cells treated with API, DMSO, and the DNMT/HDAC inhibitor (5-aza/TSA)

Techniques Used: Expressing

API Significantly Decreases the Binding of Anti-methyl Cytosine Antibody to the 15 CpG Sites in the Nrf2 Promoter in JB6 P + Cells
Figure Legend Snippet: API Significantly Decreases the Binding of Anti-methyl Cytosine Antibody to the 15 CpG Sites in the Nrf2 Promoter in JB6 P + Cells

Techniques Used: Binding Assay

69) Product Images from "Trimetazidine Attenuates Cardiac Dysfunction in Endotoxemia and Sepsis by Promoting Neutrophil Migration"

Article Title: Trimetazidine Attenuates Cardiac Dysfunction in Endotoxemia and Sepsis by Promoting Neutrophil Migration

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02015

CXCR2 is transcriptionally regulated by TMZ via Nrf2 in LPS-induced cardiac dysfunction. (A) Nrf2 ARE consensus binding site, potential Nrf2 binding site and the mutant Nrf2 binding site relative to potential Nrf2 binding site on mouse CXCR2. Blue indicates binding site, red indicates mutated base. (B) Luciferase activity of the constructs transfected into HEK293T cells. The first base before ATG represents−1. (C) Nrf2 chromatin immunoprecipitation. HEK293T cells were transfected with an empty plasmid or plasmid expressing Nrf2. PCR assays on input and IP fractions amplified the CXCR2 promoter containing the putative Nrf2 site (−919/−909, top panel) or a distal region of the Nrf2 promoter (−2727/−2717, bottom panel). (D) −1408/+254 CXCR2 promoter constructs and si-Nrf2 were transfected into HEK293T cells, and then subjected to TMZ (20 μM), LPS (5 μg/ml) or combination stimulations. Relative luciferase activity of−1408/+254 CXCR2 promoter construct after stimulations. (E) Schematic drawing of experimental schedule for the mice study. 8–10 week-old C57BL/6 male mice were first intraperitoneally injected with the CXCR2 inhibitor SB225002 (10 mg/kg) and LPS (15 mg/kg), then TMZ was administrated by gavage every 6 h for 3 times after LPS injection for 6 h. (F,G) Ejection fraction and fractional shortening was measured by two-dimensional echocardiography. (H) Representative images of left ventricular myocardium H E staining. Scale bars: 100 μm. Data is presented as mean ± SEM in vivo and mean ± SD in vitro of three independent experiments. * P
Figure Legend Snippet: CXCR2 is transcriptionally regulated by TMZ via Nrf2 in LPS-induced cardiac dysfunction. (A) Nrf2 ARE consensus binding site, potential Nrf2 binding site and the mutant Nrf2 binding site relative to potential Nrf2 binding site on mouse CXCR2. Blue indicates binding site, red indicates mutated base. (B) Luciferase activity of the constructs transfected into HEK293T cells. The first base before ATG represents−1. (C) Nrf2 chromatin immunoprecipitation. HEK293T cells were transfected with an empty plasmid or plasmid expressing Nrf2. PCR assays on input and IP fractions amplified the CXCR2 promoter containing the putative Nrf2 site (−919/−909, top panel) or a distal region of the Nrf2 promoter (−2727/−2717, bottom panel). (D) −1408/+254 CXCR2 promoter constructs and si-Nrf2 were transfected into HEK293T cells, and then subjected to TMZ (20 μM), LPS (5 μg/ml) or combination stimulations. Relative luciferase activity of−1408/+254 CXCR2 promoter construct after stimulations. (E) Schematic drawing of experimental schedule for the mice study. 8–10 week-old C57BL/6 male mice were first intraperitoneally injected with the CXCR2 inhibitor SB225002 (10 mg/kg) and LPS (15 mg/kg), then TMZ was administrated by gavage every 6 h for 3 times after LPS injection for 6 h. (F,G) Ejection fraction and fractional shortening was measured by two-dimensional echocardiography. (H) Representative images of left ventricular myocardium H E staining. Scale bars: 100 μm. Data is presented as mean ± SEM in vivo and mean ± SD in vitro of three independent experiments. * P

Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Construct, Transfection, Chromatin Immunoprecipitation, Plasmid Preparation, Expressing, Polymerase Chain Reaction, Amplification, Mouse Assay, Injection, Staining, In Vivo, In Vitro

TMZ promoted neutrophil migration by decreasing GRK2 and increasing CXCR2 expression in an AMPK/Nrf2 dependent manner. (A) BMDNs were first treated with CC (1 μM) for 1 h, then subjected to LPS stimulation (5 μg/ml) for 1 h, subsequently treated with TMZ (20 μM) for 2 h. Western blotting analysis of nuclear Nrf2 and total GRK2 in response to different stimulations. Lamin B and GAPDH were used as loading controls, respectively. (B,C) Quantification of Nrf2/Lamin B and GRK2/GAPDH was performed from the western blotting and expressed as fold induction. (D) BMDNs were first transfected with si-Nrf2 for 24 h, then subjected to LPS stimulation (5 μg/ml) for 1 h, subsequently treated with TMZ (20 μM) for 2 h. Representative images of transwell assays for BMDNs under optical microscope. Scale bar: 50 μm. (E) Relative quantitative assay of migrating BMDNs under optical microscopy. (F) Flow cytometry was performed to examine the expression of CXCR2 on the membrane of BMDNs. (G) Quantitative assay of CXCR2 expression by FACS. (H) Western blotting analysis of nuclear Nrf2 and total GRK2 in response to different stimulations after transfection. Lamin B and GAPDH were used as loading controls, respectively. (I,J) Quantification of Nrf2/Lamin B and GRK2/GAPDH was performed from the western blotting and expressed as fold induction. Data is presented as mean ± SEM in vivo and mean ± SD in vitro of three independent experiments. * P
Figure Legend Snippet: TMZ promoted neutrophil migration by decreasing GRK2 and increasing CXCR2 expression in an AMPK/Nrf2 dependent manner. (A) BMDNs were first treated with CC (1 μM) for 1 h, then subjected to LPS stimulation (5 μg/ml) for 1 h, subsequently treated with TMZ (20 μM) for 2 h. Western blotting analysis of nuclear Nrf2 and total GRK2 in response to different stimulations. Lamin B and GAPDH were used as loading controls, respectively. (B,C) Quantification of Nrf2/Lamin B and GRK2/GAPDH was performed from the western blotting and expressed as fold induction. (D) BMDNs were first transfected with si-Nrf2 for 24 h, then subjected to LPS stimulation (5 μg/ml) for 1 h, subsequently treated with TMZ (20 μM) for 2 h. Representative images of transwell assays for BMDNs under optical microscope. Scale bar: 50 μm. (E) Relative quantitative assay of migrating BMDNs under optical microscopy. (F) Flow cytometry was performed to examine the expression of CXCR2 on the membrane of BMDNs. (G) Quantitative assay of CXCR2 expression by FACS. (H) Western blotting analysis of nuclear Nrf2 and total GRK2 in response to different stimulations after transfection. Lamin B and GAPDH were used as loading controls, respectively. (I,J) Quantification of Nrf2/Lamin B and GRK2/GAPDH was performed from the western blotting and expressed as fold induction. Data is presented as mean ± SEM in vivo and mean ± SD in vitro of three independent experiments. * P

Techniques Used: Migration, Expressing, Western Blot, Transfection, Microscopy, Flow Cytometry, Cytometry, FACS, In Vivo, In Vitro

70) Product Images from "Transforming Growth Factor-? Induces Transcription Factors MafK and Bach1 to Suppress Expression of the Heme Oxygenase-1 Gene *"

Article Title: Transforming Growth Factor-? Induces Transcription Factors MafK and Bach1 to Suppress Expression of the Heme Oxygenase-1 Gene *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.450478

MafK and MafG, but not MafF, suppress transcriptional activity of HO-1 . A , pHO1-luc reporter activities were activated by overexpression of Nrf2, and the effects of MafF, MafG, and MafK were examined. Error bars represent S.D. B , interaction between Nrf2 and small Mafs was examined by coprecipitation assays in 293T cells. MafF, MafG, and MafK all coprecipitated Nrf2. C , binding of small Mafs to AREs from HO-1 . HA-tagged MafF, MafG, and MafK were expressed in 293T cells, and the cell lysates were incubated with biotinylated double-stranded DNA fragments ( Table 3 ) and precipitated with avidin beads. Coprecipitated proteins were detected with anti-HA antibody. D , chromatin immunoprecipitation analysis using anti-FLAG antibody detected binding of MafK and MafG to the HO-1 promoter region including ARE (E2) in NMuMG-MafK ( K ) and NMuMG-MafG ( G ) cells, respectively. DNAP , DNA affinity precipitation; IP , immunoprecipitation.
Figure Legend Snippet: MafK and MafG, but not MafF, suppress transcriptional activity of HO-1 . A , pHO1-luc reporter activities were activated by overexpression of Nrf2, and the effects of MafF, MafG, and MafK were examined. Error bars represent S.D. B , interaction between Nrf2 and small Mafs was examined by coprecipitation assays in 293T cells. MafF, MafG, and MafK all coprecipitated Nrf2. C , binding of small Mafs to AREs from HO-1 . HA-tagged MafF, MafG, and MafK were expressed in 293T cells, and the cell lysates were incubated with biotinylated double-stranded DNA fragments ( Table 3 ) and precipitated with avidin beads. Coprecipitated proteins were detected with anti-HA antibody. D , chromatin immunoprecipitation analysis using anti-FLAG antibody detected binding of MafK and MafG to the HO-1 promoter region including ARE (E2) in NMuMG-MafK ( K ) and NMuMG-MafG ( G ) cells, respectively. DNAP , DNA affinity precipitation; IP , immunoprecipitation.

Techniques Used: Activity Assay, Over Expression, Binding Assay, Incubation, Avidin-Biotin Assay, Chromatin Immunoprecipitation, Affinity Precipitation, Immunoprecipitation

TGF-β does not affect the stabilization and nuclear accumulation of Nrf2. A , NMuMG cells were treated with TGF-β (5 ng/ml) for 1 h before stimulation with t BHQ (25 μ m ) and incubated for an additional 4 h. Immunoblot analysis was performed using anti-Nrf2, -phospho-Smad2 ( P-Smad2 ), -Smad2 and -α-tubulin antibodies as indicated. Arrow , specific band for Nrf2; *, nonspecific bands. B , NMuMG cells were treated as in A . After fixation, cells were serially stained with anti-Nrf2 ( green ) and anti-Smad2 ( red ) antibodies. Nuclei were counterstained with DAPI. Scale bar , 50 μm. C , NMuMG cells were treated as in A . Nuclear and cytosolic fractions were isolated and analyzed by immunoblotting using antibodies for Nrf2 and Smad2. Lamin A/C was used as a nuclear protein marker, and α-tubulin was used as a cytosolic protein marker.
Figure Legend Snippet: TGF-β does not affect the stabilization and nuclear accumulation of Nrf2. A , NMuMG cells were treated with TGF-β (5 ng/ml) for 1 h before stimulation with t BHQ (25 μ m ) and incubated for an additional 4 h. Immunoblot analysis was performed using anti-Nrf2, -phospho-Smad2 ( P-Smad2 ), -Smad2 and -α-tubulin antibodies as indicated. Arrow , specific band for Nrf2; *, nonspecific bands. B , NMuMG cells were treated as in A . After fixation, cells were serially stained with anti-Nrf2 ( green ) and anti-Smad2 ( red ) antibodies. Nuclei were counterstained with DAPI. Scale bar , 50 μm. C , NMuMG cells were treated as in A . Nuclear and cytosolic fractions were isolated and analyzed by immunoblotting using antibodies for Nrf2 and Smad2. Lamin A/C was used as a nuclear protein marker, and α-tubulin was used as a cytosolic protein marker.

Techniques Used: Incubation, Staining, Isolation, Marker

Effects of TGF-β on the recruitment of Nrf2, MafK, and Bach1 to AREs (E1 and E2) in the HO-1 promoter. NMuMG cells were treated with TGF-β (5 ng/ml) for 1 h before stimulation with t BHQ (25 μ m ) for 4 h. After fixation, soluble chromatin was immunoprecipitated using anti-Nrf2 ( A ), anti-MafK ( B ), or anti-Bach1 ( C ) antibody as indicated. HO-1 promoter fragments containing AREs (E1 and E2) were amplified by PCR. Input , total chromatin solution analyzed as a control.
Figure Legend Snippet: Effects of TGF-β on the recruitment of Nrf2, MafK, and Bach1 to AREs (E1 and E2) in the HO-1 promoter. NMuMG cells were treated with TGF-β (5 ng/ml) for 1 h before stimulation with t BHQ (25 μ m ) for 4 h. After fixation, soluble chromatin was immunoprecipitated using anti-Nrf2 ( A ), anti-MafK ( B ), or anti-Bach1 ( C ) antibody as indicated. HO-1 promoter fragments containing AREs (E1 and E2) were amplified by PCR. Input , total chromatin solution analyzed as a control.

Techniques Used: Immunoprecipitation, Amplification, Polymerase Chain Reaction

71) Product Images from "Transforming Growth Factor-? Induces Transcription Factors MafK and Bach1 to Suppress Expression of the Heme Oxygenase-1 Gene *"

Article Title: Transforming Growth Factor-? Induces Transcription Factors MafK and Bach1 to Suppress Expression of the Heme Oxygenase-1 Gene *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.450478

MafK and MafG, but not MafF, suppress transcriptional activity of HO-1 . A , pHO1-luc reporter activities were activated by overexpression of Nrf2, and the effects of MafF, MafG, and MafK were examined. Error bars represent S.D. B , interaction between Nrf2 and small Mafs was examined by coprecipitation assays in 293T cells. MafF, MafG, and MafK all coprecipitated Nrf2. C , binding of small Mafs to AREs from HO-1 . HA-tagged MafF, MafG, and MafK were expressed in 293T cells, and the cell lysates were incubated with biotinylated double-stranded DNA fragments ( Table 3 ) and precipitated with avidin beads. Coprecipitated proteins were detected with anti-HA antibody. D , chromatin immunoprecipitation analysis using anti-FLAG antibody detected binding of MafK and MafG to the HO-1 promoter region including ARE (E2) in NMuMG-MafK ( K ) and NMuMG-MafG ( G ) cells, respectively. DNAP , DNA affinity precipitation; IP , immunoprecipitation.
Figure Legend Snippet: MafK and MafG, but not MafF, suppress transcriptional activity of HO-1 . A , pHO1-luc reporter activities were activated by overexpression of Nrf2, and the effects of MafF, MafG, and MafK were examined. Error bars represent S.D. B , interaction between Nrf2 and small Mafs was examined by coprecipitation assays in 293T cells. MafF, MafG, and MafK all coprecipitated Nrf2. C , binding of small Mafs to AREs from HO-1 . HA-tagged MafF, MafG, and MafK were expressed in 293T cells, and the cell lysates were incubated with biotinylated double-stranded DNA fragments ( Table 3 ) and precipitated with avidin beads. Coprecipitated proteins were detected with anti-HA antibody. D , chromatin immunoprecipitation analysis using anti-FLAG antibody detected binding of MafK and MafG to the HO-1 promoter region including ARE (E2) in NMuMG-MafK ( K ) and NMuMG-MafG ( G ) cells, respectively. DNAP , DNA affinity precipitation; IP , immunoprecipitation.

Techniques Used: Activity Assay, Over Expression, Binding Assay, Incubation, Avidin-Biotin Assay, Chromatin Immunoprecipitation, Affinity Precipitation, Immunoprecipitation

TGF-β does not affect the stabilization and nuclear accumulation of Nrf2. A , NMuMG cells were treated with TGF-β (5 ng/ml) for 1 h before stimulation with t BHQ (25 μ m ) and incubated for an additional 4 h. Immunoblot analysis was performed using anti-Nrf2, -phospho-Smad2 ( P-Smad2 ), -Smad2 and -α-tubulin antibodies as indicated. Arrow , specific band for Nrf2; *, nonspecific bands. B , NMuMG cells were treated as in A . After fixation, cells were serially stained with anti-Nrf2 ( green ) and anti-Smad2 ( red ) antibodies. Nuclei were counterstained with DAPI. Scale bar , 50 μm. C , NMuMG cells were treated as in A . Nuclear and cytosolic fractions were isolated and analyzed by immunoblotting using antibodies for Nrf2 and Smad2. Lamin A/C was used as a nuclear protein marker, and α-tubulin was used as a cytosolic protein marker.
Figure Legend Snippet: TGF-β does not affect the stabilization and nuclear accumulation of Nrf2. A , NMuMG cells were treated with TGF-β (5 ng/ml) for 1 h before stimulation with t BHQ (25 μ m ) and incubated for an additional 4 h. Immunoblot analysis was performed using anti-Nrf2, -phospho-Smad2 ( P-Smad2 ), -Smad2 and -α-tubulin antibodies as indicated. Arrow , specific band for Nrf2; *, nonspecific bands. B , NMuMG cells were treated as in A . After fixation, cells were serially stained with anti-Nrf2 ( green ) and anti-Smad2 ( red ) antibodies. Nuclei were counterstained with DAPI. Scale bar , 50 μm. C , NMuMG cells were treated as in A . Nuclear and cytosolic fractions were isolated and analyzed by immunoblotting using antibodies for Nrf2 and Smad2. Lamin A/C was used as a nuclear protein marker, and α-tubulin was used as a cytosolic protein marker.

Techniques Used: Incubation, Staining, Isolation, Marker

Effects of TGF-β on the recruitment of Nrf2, MafK, and Bach1 to AREs (E1 and E2) in the HO-1 promoter. NMuMG cells were treated with TGF-β (5 ng/ml) for 1 h before stimulation with t BHQ (25 μ m ) for 4 h. After fixation, soluble chromatin was immunoprecipitated using anti-Nrf2 ( A ), anti-MafK ( B ), or anti-Bach1 ( C ) antibody as indicated. HO-1 promoter fragments containing AREs (E1 and E2) were amplified by PCR. Input , total chromatin solution analyzed as a control.
Figure Legend Snippet: Effects of TGF-β on the recruitment of Nrf2, MafK, and Bach1 to AREs (E1 and E2) in the HO-1 promoter. NMuMG cells were treated with TGF-β (5 ng/ml) for 1 h before stimulation with t BHQ (25 μ m ) for 4 h. After fixation, soluble chromatin was immunoprecipitated using anti-Nrf2 ( A ), anti-MafK ( B ), or anti-Bach1 ( C ) antibody as indicated. HO-1 promoter fragments containing AREs (E1 and E2) were amplified by PCR. Input , total chromatin solution analyzed as a control.

Techniques Used: Immunoprecipitation, Amplification, Polymerase Chain Reaction

72) 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

73) Product Images from "Modulation of renal superoxide dismutase by telmisartan therapy in C57BL/6-Ins2Akita diabetic mice"

Article Title: Modulation of renal superoxide dismutase by telmisartan therapy in C57BL/6-Ins2Akita diabetic mice

Journal: Hypertension Research

doi: 10.1038/hr.2011.176

Western blot analysis of renal cortical SOD isoform and Nrf2 expression after an 8-week treatment with the vehicle (VE) or apocynin (AP) in C57BL/6-Akita diabetic mice. WT indicates non-diabetic C57BL/6-wild-type mice. The relative intensity of the SOD-to-actin or Nrf2-to-actin ratios to WT is also shown in the lower panels. Data are presented as the mean±s.e.m. n =4 per group. * P
Figure Legend Snippet: Western blot analysis of renal cortical SOD isoform and Nrf2 expression after an 8-week treatment with the vehicle (VE) or apocynin (AP) in C57BL/6-Akita diabetic mice. WT indicates non-diabetic C57BL/6-wild-type mice. The relative intensity of the SOD-to-actin or Nrf2-to-actin ratios to WT is also shown in the lower panels. Data are presented as the mean±s.e.m. n =4 per group. * P

Techniques Used: Western Blot, Expressing, Mouse Assay

Western blot analysis of renal cortical SOD isoforms and Nrf2 expression after a 4-week treatment with the vehicle (VE), amlodipine (AM) or telmisartan (TE) in C57BL/6-Akita diabetic mice. WT indicates non-diabetic C57BL/6-wild-type mice. The relative intensity of the SOD-to-actin or Nrf2-to-actin ratios to WT is also shown in the lower panels. Data are presented as the mean±s.e.m. n =4 per group. * P
Figure Legend Snippet: Western blot analysis of renal cortical SOD isoforms and Nrf2 expression after a 4-week treatment with the vehicle (VE), amlodipine (AM) or telmisartan (TE) in C57BL/6-Akita diabetic mice. WT indicates non-diabetic C57BL/6-wild-type mice. The relative intensity of the SOD-to-actin or Nrf2-to-actin ratios to WT is also shown in the lower panels. Data are presented as the mean±s.e.m. n =4 per group. * P

Techniques Used: Western Blot, Expressing, Mouse Assay

74) Product Images from "The decay of Redox-stress Response Capacity is a substantive characteristic of aging: Revising the redox theory of aging"

Article Title: The decay of Redox-stress Response Capacity is a substantive characteristic of aging: Revising the redox theory of aging

Journal: Redox Biology

doi: 10.1016/j.redox.2016.12.026

Young individuals possess a stronger ability to maintain redox homeostasis. (A) The relative expression of sod1 , sod2 , sod3 and gcs-1 in Day 1 and Day 12 worms treated with PQ was assessed by performing qRT-PCR. (B) The relative expression of skn-1 in Day 1 and Day 12 worms treated with PQ was assessed by performing qRT-PCR. (C) NRF2 expression in p34 and p42 cells in response to PQ-induced stress was assessed by performing Western blotting at the indicated times. NRF2 levels were normalized to Actin. (E) NRF2 localization was examined by performing immunofluorescence staining on cells that were treated with PQ for 24 h. (F) The expression of SOD1 , CAT , GPX and TR1 in p34 and p42 cells treated with PQ was detected by qRT-PCR at the indicated times. (G) Total SOD and CAT enzyme activities in p34 and p42 cells were measured at 24 h and 48 h after stress using commercial kits. (H) Expression of the phase II enzymes HO-1 and GSTP1 in p34 and p42 cells treated with PQ was determined by performing qRT-PCR at the indicated times. The error bars indicate the SEM (n=3).
Figure Legend Snippet: Young individuals possess a stronger ability to maintain redox homeostasis. (A) The relative expression of sod1 , sod2 , sod3 and gcs-1 in Day 1 and Day 12 worms treated with PQ was assessed by performing qRT-PCR. (B) The relative expression of skn-1 in Day 1 and Day 12 worms treated with PQ was assessed by performing qRT-PCR. (C) NRF2 expression in p34 and p42 cells in response to PQ-induced stress was assessed by performing Western blotting at the indicated times. NRF2 levels were normalized to Actin. (E) NRF2 localization was examined by performing immunofluorescence staining on cells that were treated with PQ for 24 h. (F) The expression of SOD1 , CAT , GPX and TR1 in p34 and p42 cells treated with PQ was detected by qRT-PCR at the indicated times. (G) Total SOD and CAT enzyme activities in p34 and p42 cells were measured at 24 h and 48 h after stress using commercial kits. (H) Expression of the phase II enzymes HO-1 and GSTP1 in p34 and p42 cells treated with PQ was determined by performing qRT-PCR at the indicated times. The error bars indicate the SEM (n=3).

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

75) Product Images from "Biochanin A Provides Neuroprotection Against Cerebral Ischemia/Reperfusion Injury by Nrf2-Mediated Inhibition of Oxidative Stress and Inflammation Signaling Pathway in Rats"

Article Title: Biochanin A Provides Neuroprotection Against Cerebral Ischemia/Reperfusion Injury by Nrf2-Mediated Inhibition of Oxidative Stress and Inflammation Signaling Pathway in Rats

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.918665

Biochanin A induced Nrf2 nuclear translocation in cerebral I/R rats. Protein extractions in the cytosolic and nuclear fractions from the ischemic penumbras zone of rat brain tissues were isolated, and the expression of Nrf2 was determined by Western blotting at 24 h after reperfusion. Representative Western blots and protein expression of Nrf2 in the cytosolic fractions ( A ) and nuclear fractions ( B ) were analyzed. α-Tubulin and PCNA were used as a loading control in cytosolic protein and in nuclear protein. The protein expression of Nrf2 were compared between the sham group and the I/R group ( ## P
Figure Legend Snippet: Biochanin A induced Nrf2 nuclear translocation in cerebral I/R rats. Protein extractions in the cytosolic and nuclear fractions from the ischemic penumbras zone of rat brain tissues were isolated, and the expression of Nrf2 was determined by Western blotting at 24 h after reperfusion. Representative Western blots and protein expression of Nrf2 in the cytosolic fractions ( A ) and nuclear fractions ( B ) were analyzed. α-Tubulin and PCNA were used as a loading control in cytosolic protein and in nuclear protein. The protein expression of Nrf2 were compared between the sham group and the I/R group ( ## P

Techniques Used: Translocation Assay, Isolation, Expressing, Western Blot

Related Articles

Clone Assay:

Article Title: The bromodomain protein BRD4 regulates the KEAP1/NRF2-dependent oxidative stress response
Article Snippet: The enhancer region E1 was cloned into the BamH1 and SalI restriction sites in the pGL3-basic vector using the primers HMOX1-E1fw: 5′-ACAATTGGCCCAGTCTATGG-3′ and HMOX1-E1rev: 5′-GGAGTTCAAGACCAGCCTGA-3′. .. The following antibodies were used: rabbit polyclonal BRD4 antibody (ab75898, Abcam, Cambridge, UK), mouse monoclonal HMOX1 antibody (ab13243, Abcam), NRF2 (sc13032, sc722, Santa Cruz Biotechnology, Heidelberg, Germany), KEAP1 (K2769, Sigma-Aldrich, Taufkirchen, Germany), alpha-tubulin (T9026, Sigma-Aldrich), and beta-actin (Cell Signaling, Leiden, Netherlands).

Article Title: Hepatitis B virus stimulates G6PD expression through HBx-mediated Nrf2 activation
Article Snippet: Antibodies, reagents, and plasmids The following antibodies were used: anti-β -actin, anti-G6PD (Sigma-Aldrich); anti-HBcAg (Abcam, Cambridge, MA, USA), anti-Keap1 (Cell Signaling, Danvers, MA, USA); anti-Myc, anti-Nrf2 (sc-365949), anti-Lamin B, anti-p62, and anti-Flag (Santa Cruz, Santa Cruz, CA, USA); anti-GFP (BD Biosciences, Franklin Lakes, NJ, USA); goat anti-rabbit IRDye800CW and goat anti-mouse IRDye680 (LI-COR Biosciences, Lincoln, NE, USA). .. HBx-Myc was made by cloning the cDNA of HBx into pcDNA-3.1 vector using Eco RI and Xho I restriction sites.

Luciferase:

Article Title: The bromodomain protein BRD4 regulates the KEAP1/NRF2-dependent oxidative stress response
Article Snippet: For the luciferase reporter assay, the HMOX1 promoter was cloned into the pGL3-basic vector from Promega (Mannheim, Germany). .. The following antibodies were used: rabbit polyclonal BRD4 antibody (ab75898, Abcam, Cambridge, UK), mouse monoclonal HMOX1 antibody (ab13243, Abcam), NRF2 (sc13032, sc722, Santa Cruz Biotechnology, Heidelberg, Germany), KEAP1 (K2769, Sigma-Aldrich, Taufkirchen, Germany), alpha-tubulin (T9026, Sigma-Aldrich), and beta-actin (Cell Signaling, Leiden, Netherlands).

Reporter Assay:

Article Title: The bromodomain protein BRD4 regulates the KEAP1/NRF2-dependent oxidative stress response
Article Snippet: For the luciferase reporter assay, the HMOX1 promoter was cloned into the pGL3-basic vector from Promega (Mannheim, Germany). .. The following antibodies were used: rabbit polyclonal BRD4 antibody (ab75898, Abcam, Cambridge, UK), mouse monoclonal HMOX1 antibody (ab13243, Abcam), NRF2 (sc13032, sc722, Santa Cruz Biotechnology, Heidelberg, Germany), KEAP1 (K2769, Sigma-Aldrich, Taufkirchen, Germany), alpha-tubulin (T9026, Sigma-Aldrich), and beta-actin (Cell Signaling, Leiden, Netherlands).

Synthesized:

Article Title: Antitumor agent PX-12 inhibits HIF-1α protein levels through an Nrf2/PMF-1-mediated increase in spermidine/spermine acetyl transferase
Article Snippet: Monoclonal antibodies to human HIF-1 α , RACK1, and elongin C were purchased from BD Transduction Laboratories (San Diego, CA), and antibodies to Trx-1, Trx-2, Trx-reductase-1 and -2, lamin A/C, β -actin, NAD(P)H dehydrogenase, quinone 1 (NQO1), and Nrf2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). .. PX-12 was synthesized [ ], and MG-132 was purchased from Sigma–Aldrich (St. Louis, MO).

Blocking Assay:

Article Title: Metabolic Responses in Endothelial Cells Following Exposure to Ketone Bodies
Article Snippet: Then, the PVDF membranes were incubated in Tris-buffered Saline with 0.1% ( v / v ) Tween 20 (TBS-T) containing 5% ( w / v ) non-fat dried milk or 5% ( w / v ) bovine serum albumin (BSA; Sigma) for 1 h. Blots were incubated with primary antibodies in the appropriate blocking solution at 4 °C overnight. .. The following primary antibodies were used: Nrf2 (C-20, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) dilution 1:400 in TBS-T+5% ( w / v ) non-fat dried milk; GAPDH (FL-335, Santa Cruz Biotechnology, Inc.) dilution 1:500 in TBS-T+5% ( w / v ) non-fat dried milk; lamin A/C (N-18, Santa Cruz Biotechnology, Inc.) dilution 1:500 in TBS-T+5% BSA.

SYBR Green Assay:

Article Title: Heme regulates gene expression by triggering Crm1-dependent nuclear export of Bach1
Article Snippet: After 36 h, cells were treated with or without hemin for another 2 h and processed for ChIP as described previously ( ) using anti-Bach1 (A1–6), anti-Nrf2 (Santa Cruz Biotechnology, sc-13032), or anti-MafK ( ) antibodies. .. After 36 h, cells were treated with or without hemin for another 2 h and processed for ChIP as described previously ( ) using anti-Bach1 (A1–6), anti-Nrf2 (Santa Cruz Biotechnology, sc-13032), or anti-MafK ( ) antibodies.

Incubation:

Article Title: An Autoregulatory Loop between Nrf2 and Cul3-Rbx1 Controls Their Cellular Abundance *
Article Snippet: .. The membranes were incubated with anti-Cul3 (1:1000, Cell Signaling), anti-Rbx1 (1:1000, Bio Source) anti-INrf2 (1:1000, Santa Cruz Biotechnology), anti-Nrf2 (1:500, Santa Cruz Biotechnology), anti-FLAG (1:5000, Sigma), anti-V5 (1:5000, Invitrogen), anti-Myc (1:5000, Sigma), or anti-actin (1:5000, Sigma) antibodies, washed, and probed with electrochemiluminescence (Amersham Biosciences). .. To confirm the purity of nuclear-cytoplasmic fractionation, the membranes were reprobed with cytoplasm-specific, anti-lactate dehydrogenase (Chemicon) and nuclear specific, anti-lamin B antibodies (Santa Cruz Biotechnology).

Article Title: Metabolic Responses in Endothelial Cells Following Exposure to Ketone Bodies
Article Snippet: Then, the PVDF membranes were incubated in Tris-buffered Saline with 0.1% ( v / v ) Tween 20 (TBS-T) containing 5% ( w / v ) non-fat dried milk or 5% ( w / v ) bovine serum albumin (BSA; Sigma) for 1 h. Blots were incubated with primary antibodies in the appropriate blocking solution at 4 °C overnight. .. The following primary antibodies were used: Nrf2 (C-20, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) dilution 1:400 in TBS-T+5% ( w / v ) non-fat dried milk; GAPDH (FL-335, Santa Cruz Biotechnology, Inc.) dilution 1:500 in TBS-T+5% ( w / v ) non-fat dried milk; lamin A/C (N-18, Santa Cruz Biotechnology, Inc.) dilution 1:500 in TBS-T+5% BSA.

Article Title: Stress Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas
Article Snippet: .. Cell lysates were sonicated and incubated with the specific antibodies overnight: NRF2 (Santa Cruz, sc-722), MafF/G/K (H-100) (Santa Cruz, sc-22831). ..

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: Role of NADPH oxidase
Article Snippet: Then, the membranes were probed with the following primary antibodies overnight at 4 °C: anti-α-SMA (1:1000, Abcam, Cambridge, MA), anti-desmin (1:500, BD Pharmingen, San Jose, CA), anti-CB1 (1:1000, Thermo, Waltham, MA), anti-CB2 (1:1000, Abcam), anti-TIMP-1 (1:500, Santa Cruz, Dallas, TX), anti-collagen I (1:1000, Calbiochem, Billerica, MA), anti-Nox1 (1:500, Abcam), anti-Nox4 (1:500, Santa Cruz), anti-Nrf2 (1:1000, Santa Cruz), and anti-β-actin (1:10000, Santa Cruz). .. After washing with TBST three times, membranes were incubated with the following secondary antibodies for 1 h at room temperature, respectively with corresponding goat anti-rabbit-HRP or rabbit anti-mouse-HRP (1:10000, Santa Cruz) based on animals used for raising primary antibody.

Article Title: Phloretin Attenuates Allergic Airway Inflammation and Oxidative Stress in Asthmatic Mice
Article Snippet: The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and incubated with primary antibodies overnight at 4°C. .. Primary antibodies included anti-HO-1, anti-Nrf2, and anti-Lamin B1 (Santa Cruz, CA, USA); β-actin expression was evaluated as a loading control (Sigma).

Amplification:

Article Title: Hepatitis B virus stimulates G6PD expression through HBx-mediated Nrf2 activation
Article Snippet: Antibodies, reagents, and plasmids The following antibodies were used: anti-β -actin, anti-G6PD (Sigma-Aldrich); anti-HBcAg (Abcam, Cambridge, MA, USA), anti-Keap1 (Cell Signaling, Danvers, MA, USA); anti-Myc, anti-Nrf2 (sc-365949), anti-Lamin B, anti-p62, and anti-Flag (Santa Cruz, Santa Cruz, CA, USA); anti-GFP (BD Biosciences, Franklin Lakes, NJ, USA); goat anti-rabbit IRDye800CW and goat anti-mouse IRDye680 (LI-COR Biosciences, Lincoln, NE, USA). .. For Flag-p62 construction, the p62 coding region was amplified from Cherry-GFP-p62, a gift from Terje Johansen (University of Tromsø, Tromsø, Norway), and was inserted into pCMV-Tag 2A vector using the Eco RI and Xho I restriction sites.

Proliferation Assay:

Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells
Article Snippet: TACS MTT Cell Proliferation Assay Kit and GPX enzyme-linked immunosorbent assay (ELISA) were purchased from R & D Systems (Minneapolis, MN, USA). .. Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Expressing:

Article Title: Heme regulates gene expression by triggering Crm1-dependent nuclear export of Bach1
Article Snippet: Mouse HO-1 gene reporter plasmid pHO15luc ( ) and various combinations of expression plasmids were transfected using Fugene6 (Roche) to GM02063 cells. .. After 36 h, cells were treated with or without hemin for another 2 h and processed for ChIP as described previously ( ) using anti-Bach1 (A1–6), anti-Nrf2 (Santa Cruz Biotechnology, sc-13032), or anti-MafK ( ) antibodies.

Article Title: An Autoregulatory Loop between Nrf2 and Cul3-Rbx1 Controls Their Cellular Abundance *
Article Snippet: The membranes were incubated with anti-Cul3 (1:1000, Cell Signaling), anti-Rbx1 (1:1000, Bio Source) anti-INrf2 (1:1000, Santa Cruz Biotechnology), anti-Nrf2 (1:500, Santa Cruz Biotechnology), anti-FLAG (1:5000, Sigma), anti-V5 (1:5000, Invitrogen), anti-Myc (1:5000, Sigma), or anti-actin (1:5000, Sigma) antibodies, washed, and probed with electrochemiluminescence (Amersham Biosciences). .. Protein expression was quantified by using NIH Image program (developed at the National Institutes of Health).

Article Title: Phloretin Attenuates Allergic Airway Inflammation and Oxidative Stress in Asthmatic Mice
Article Snippet: .. Primary antibodies included anti-HO-1, anti-Nrf2, and anti-Lamin B1 (Santa Cruz, CA, USA); β-actin expression was evaluated as a loading control (Sigma). ..

Modification:

Article Title: Sinomenine Regulates Inflammatory Response and Oxidative Stress via Nuclear Factor kappa B (NF-κB) and NF-E2-Related Factor 2 (Nrf2) Signaling Pathways in Ankle Fractures in Children
Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), streptomycin, penicillin, glutamine, and sodium pyruvate were obtained from Gibco/Invitrogen, Inc. (Carlsbad, CA, USA). .. We obtained anti-Nrf2 from Santa Cruz Biotechnology (USA), anti-HO-1 was purchased from Enzo Life Sciences (USA), and anti-NQO-1 was obtained from Novus biological (USA).

Article Title: Extraction, identification, and antioxidant property evaluation of limonin from pummelo seeds
Article Snippet: The HepG2 cells were purchased from ATCC (VA, USA) and were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) plus 100 units of penicillin and streptomycin. .. The antibodies of anti-Nrf2, anti-NQO1, anti-HO-1, anti-GAPDH, and anti-Keap1 were procured from Santa Cruz Biotechnology Inc. (CA, USA).

Western Blot:

Article Title: C66 ameliorates diabetic nephropathy in mice by both upregulating NRF2 function via increase in miR-200a and inhibiting miR-21
Article Snippet: Paragraph title: Western blot analysis ... The primary antibodies were anti-KEAP1 (Santa Cruz Biotechnology, Dallas, TX, USA; 1:1,000), anti-NRF2 (Santa Cruz Biotechnology; 1:1,000), anti-Histone H3 (Santa Cruz Biotechnology; 1:500), anti-4-HNE (Alpha Diagnostic, San Antonio, TX, USA; 1:3, 000), anti-3-NT (Millipore, Temecula, CA, USA; 1:1,000), anti-TGF-β1 (Cell Signaling, Beverly, MA, USA; 1:500), anti-COL4 (Abcam, Cambridge, MA, USA, 1:500), anti-FN (Santa Cruz Biotechnology; 1:500), anti-Smad7 (Santa Cruz Biotechnology; 1:1,000), anti-Smad3 (Santa Cruz Biotechnology; 1:1,000), anti-p-Smad3 (Cell Signaling; 1:500), anti-p-JNK (Cell Signaling; 1:500), anti-PDCD4 (Santa Cruz Biotechnology; 1:1,000), anti-t-JNK (Cell Signaling; 1:1,000), anti-Actin (Santa Cruz Biotechnology; 1:2,000) and anti-GAPDH (Santa Cruz Biotechnology; 1:3,000).

Article Title: An Autoregulatory Loop between Nrf2 and Cul3-Rbx1 Controls Their Cellular Abundance *
Article Snippet: Paragraph title: Western Blot Analysis ... The membranes were incubated with anti-Cul3 (1:1000, Cell Signaling), anti-Rbx1 (1:1000, Bio Source) anti-INrf2 (1:1000, Santa Cruz Biotechnology), anti-Nrf2 (1:500, Santa Cruz Biotechnology), anti-FLAG (1:5000, Sigma), anti-V5 (1:5000, Invitrogen), anti-Myc (1:5000, Sigma), or anti-actin (1:5000, Sigma) antibodies, washed, and probed with electrochemiluminescence (Amersham Biosciences).

Article Title: CDDO-Me Protects Normal Lung and Breast Epithelial Cells but Not Cancer Cells from Radiation
Article Snippet: Paragraph title: Western Blot Analysis ... The following antibodies were used: anti- HO1, -PRX1, -NQO1 (1∶1000; AbCam), anti- Nrf2 (1∶500 Santa Cruz; 1∶1000 Cell Signaling), anti-phospho-Nrf2 (1∶5000; AbCam), and anti–β-actin (1∶20,000; Sigma).

Article Title: Metabolic Responses in Endothelial Cells Following Exposure to Ketone Bodies
Article Snippet: Paragraph title: 2.6.2. Western Blot Analysis ... The following primary antibodies were used: Nrf2 (C-20, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) dilution 1:400 in TBS-T+5% ( w / v ) non-fat dried milk; GAPDH (FL-335, Santa Cruz Biotechnology, Inc.) dilution 1:500 in TBS-T+5% ( w / v ) non-fat dried milk; lamin A/C (N-18, Santa Cruz Biotechnology, Inc.) dilution 1:500 in TBS-T+5% BSA.

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: Role of NADPH oxidase
Article Snippet: Paragraph title: Western blotting ... Then, the membranes were probed with the following primary antibodies overnight at 4 °C: anti-α-SMA (1:1000, Abcam, Cambridge, MA), anti-desmin (1:500, BD Pharmingen, San Jose, CA), anti-CB1 (1:1000, Thermo, Waltham, MA), anti-CB2 (1:1000, Abcam), anti-TIMP-1 (1:500, Santa Cruz, Dallas, TX), anti-collagen I (1:1000, Calbiochem, Billerica, MA), anti-Nox1 (1:500, Abcam), anti-Nox4 (1:500, Santa Cruz), anti-Nrf2 (1:1000, Santa Cruz), and anti-β-actin (1:10000, Santa Cruz).

Article Title: Phloretin Attenuates Allergic Airway Inflammation and Oxidative Stress in Asthmatic Mice
Article Snippet: Paragraph title: Western Immunoblot Analysis ... Primary antibodies included anti-HO-1, anti-Nrf2, and anti-Lamin B1 (Santa Cruz, CA, USA); β-actin expression was evaluated as a loading control (Sigma).

Transformation Assay:

Article Title: Antitumor agent PX-12 inhibits HIF-1α protein levels through an Nrf2/PMF-1-mediated increase in spermidine/spermine acetyl transferase
Article Snippet: Human MiaPaCa-2 pancreatic cancer cells, MCF-7 breast cancer cells, LN-229 glioma cancer cells, and HEK-293 transformed embryonic kidney cells were obtained from American Type Culture Collection (Manassas, VA). .. Monoclonal antibodies to human HIF-1 α , RACK1, and elongin C were purchased from BD Transduction Laboratories (San Diego, CA), and antibodies to Trx-1, Trx-2, Trx-reductase-1 and -2, lamin A/C, β -actin, NAD(P)H dehydrogenase, quinone 1 (NQO1), and Nrf2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Transfection:

Article Title: Heme regulates gene expression by triggering Crm1-dependent nuclear export of Bach1
Article Snippet: Mouse HO-1 gene reporter plasmid pHO15luc ( ) and various combinations of expression plasmids were transfected using Fugene6 (Roche) to GM02063 cells. .. After 36 h, cells were treated with or without hemin for another 2 h and processed for ChIP as described previously ( ) using anti-Bach1 (A1–6), anti-Nrf2 (Santa Cruz Biotechnology, sc-13032), or anti-MafK ( ) antibodies.

Protease Inhibitor:

Article Title: An Autoregulatory Loop between Nrf2 and Cul3-Rbx1 Controls Their Cellular Abundance *
Article Snippet: The cells were lysed in ice-cold RIPA-B buffer (20 m m Tris, pH 7.4, 150 m m NaCl, 1 m m EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 1% Triton X-100), and protease inhibitor mixture (Roche Applied Science). .. The membranes were incubated with anti-Cul3 (1:1000, Cell Signaling), anti-Rbx1 (1:1000, Bio Source) anti-INrf2 (1:1000, Santa Cruz Biotechnology), anti-Nrf2 (1:500, Santa Cruz Biotechnology), anti-FLAG (1:5000, Sigma), anti-V5 (1:5000, Invitrogen), anti-Myc (1:5000, Sigma), or anti-actin (1:5000, Sigma) antibodies, washed, and probed with electrochemiluminescence (Amersham Biosciences).

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: Role of NADPH oxidase
Article Snippet: After various treatment, HSCs were washed twice with PBS and scraped in a lysis buffer (20 mM Tris-HCl, 250 mM sucrose, pH 7.2) containing 1X protease inhibitor mixture (Roche, Indianapolis, IN). .. Then, the membranes were probed with the following primary antibodies overnight at 4 °C: anti-α-SMA (1:1000, Abcam, Cambridge, MA), anti-desmin (1:500, BD Pharmingen, San Jose, CA), anti-CB1 (1:1000, Thermo, Waltham, MA), anti-CB2 (1:1000, Abcam), anti-TIMP-1 (1:500, Santa Cruz, Dallas, TX), anti-collagen I (1:1000, Calbiochem, Billerica, MA), anti-Nox1 (1:500, Abcam), anti-Nox4 (1:500, Santa Cruz), anti-Nrf2 (1:1000, Santa Cruz), and anti-β-actin (1:10000, Santa Cruz).

Cell Culture:

Article Title: Extraction, identification, and antioxidant property evaluation of limonin from pummelo seeds
Article Snippet: Paragraph title: 2.1. Raw material, chemicals, and cell culture ... The antibodies of anti-Nrf2, anti-NQO1, anti-HO-1, anti-GAPDH, and anti-Keap1 were procured from Santa Cruz Biotechnology Inc. (CA, USA).

Mutagenesis:

Article Title: The bromodomain protein BRD4 regulates the KEAP1/NRF2-dependent oxidative stress response
Article Snippet: Paragraph title: Plasmids, RNA interference, site-directed mutagenesis and antibodies ... The following antibodies were used: rabbit polyclonal BRD4 antibody (ab75898, Abcam, Cambridge, UK), mouse monoclonal HMOX1 antibody (ab13243, Abcam), NRF2 (sc13032, sc722, Santa Cruz Biotechnology, Heidelberg, Germany), KEAP1 (K2769, Sigma-Aldrich, Taufkirchen, Germany), alpha-tubulin (T9026, Sigma-Aldrich), and beta-actin (Cell Signaling, Leiden, Netherlands).

Generated:

Article Title: Heme regulates gene expression by triggering Crm1-dependent nuclear export of Bach1
Article Snippet: Polyclonal antiserum against Bach1 (A1–6) was generated by immunizing rabbits with a GST fusion protein of mouse Bach1 (amino-acid residues 624–739). .. After 36 h, cells were treated with or without hemin for another 2 h and processed for ChIP as described previously ( ) using anti-Bach1 (A1–6), anti-Nrf2 (Santa Cruz Biotechnology, sc-13032), or anti-MafK ( ) antibodies.

Polymerase Chain Reaction:

Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells
Article Snippet: Human Oxidative Stress PCR Array was purchased from Qiagen (Valencia, CA, USA). .. Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Sonication:

Article Title: Stress Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas
Article Snippet: .. Cell lysates were sonicated and incubated with the specific antibodies overnight: NRF2 (Santa Cruz, sc-722), MafF/G/K (H-100) (Santa Cruz, sc-22831). ..

MTT Assay:

Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells
Article Snippet: TACS MTT Cell Proliferation Assay Kit and GPX enzyme-linked immunosorbent assay (ELISA) were purchased from R & D Systems (Minneapolis, MN, USA). .. Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Article Title: Notoginsenoside R1 ameliorates diabetic encephalopathy by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation
Article Snippet: Primary antibodies against Akt, ASC, IL-1β, and Nrf2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). .. 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT), PI3K inhibitor LY294002, and all of other regents were obtained from Sigma-Aldrich (St. Louis, USA).

Raw Material:

Article Title: Extraction, identification, and antioxidant property evaluation of limonin from pummelo seeds
Article Snippet: Paragraph title: 2.1. Raw material, chemicals, and cell culture ... The antibodies of anti-Nrf2, anti-NQO1, anti-HO-1, anti-GAPDH, and anti-Keap1 were procured from Santa Cruz Biotechnology Inc. (CA, USA).

Multiple Displacement Amplification:

Article Title: Notoginsenoside R1 ameliorates diabetic encephalopathy by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation
Article Snippet: The kits for determining the lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were obtained from Nanjing jiancheng Bioengineering Institute (Nanjing, China). .. Primary antibodies against Akt, ASC, IL-1β, and Nrf2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Chromatin Immunoprecipitation:

Article Title: Heme regulates gene expression by triggering Crm1-dependent nuclear export of Bach1
Article Snippet: .. After 36 h, cells were treated with or without hemin for another 2 h and processed for ChIP as described previously ( ) using anti-Bach1 (A1–6), anti-Nrf2 (Santa Cruz Biotechnology, sc-13032), or anti-MafK ( ) antibodies. .. Amounts of precipitated DNA were analyzed in duplicate by real-time PCR (LightCycler and SYBR Green 1, Roche).

Article Title: Stress Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas
Article Snippet: Paragraph title: Chromatin-immunoprecipitation (ChIP) ... Cell lysates were sonicated and incubated with the specific antibodies overnight: NRF2 (Santa Cruz, sc-722), MafF/G/K (H-100) (Santa Cruz, sc-22831).

SDS Page:

Article Title: CDDO-Me Protects Normal Lung and Breast Epithelial Cells but Not Cancer Cells from Radiation
Article Snippet: Western Blot Analysis Cells were lysed in Laemelli SDS reducing buffer [50 mM Tris-HCl (pH 6.8), 2% SDS, and 10% glycerol], boiled, and separated by SDS/PAGE. .. The following antibodies were used: anti- HO1, -PRX1, -NQO1 (1∶1000; AbCam), anti- Nrf2 (1∶500 Santa Cruz; 1∶1000 Cell Signaling), anti-phospho-Nrf2 (1∶5000; AbCam), and anti–β-actin (1∶20,000; Sigma).

Article Title: Metabolic Responses in Endothelial Cells Following Exposure to Ketone Bodies
Article Snippet: Proteins were denatured by boiling for 5 min in sodium dodecylsulfate (SDS) sample buffer, loaded into 10% SDS-PAGE gels and subsequently transferred onto PVDF membranes by electroblotting. .. The following primary antibodies were used: Nrf2 (C-20, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) dilution 1:400 in TBS-T+5% ( w / v ) non-fat dried milk; GAPDH (FL-335, Santa Cruz Biotechnology, Inc.) dilution 1:500 in TBS-T+5% ( w / v ) non-fat dried milk; lamin A/C (N-18, Santa Cruz Biotechnology, Inc.) dilution 1:500 in TBS-T+5% BSA.

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: Role of NADPH oxidase
Article Snippet: Samples were run by SDS-PAGE, transferred onto a PVDF membrane, and blocked using 5% nonfat dry milk (NFDM) in Tris-buffered saline with 0.1% Tween 20 (TBST). .. Then, the membranes were probed with the following primary antibodies overnight at 4 °C: anti-α-SMA (1:1000, Abcam, Cambridge, MA), anti-desmin (1:500, BD Pharmingen, San Jose, CA), anti-CB1 (1:1000, Thermo, Waltham, MA), anti-CB2 (1:1000, Abcam), anti-TIMP-1 (1:500, Santa Cruz, Dallas, TX), anti-collagen I (1:1000, Calbiochem, Billerica, MA), anti-Nox1 (1:500, Abcam), anti-Nox4 (1:500, Santa Cruz), anti-Nrf2 (1:1000, Santa Cruz), and anti-β-actin (1:10000, Santa Cruz).

Plasmid Preparation:

Article Title: Heme regulates gene expression by triggering Crm1-dependent nuclear export of Bach1
Article Snippet: Mouse HO-1 gene reporter plasmid pHO15luc ( ) and various combinations of expression plasmids were transfected using Fugene6 (Roche) to GM02063 cells. .. After 36 h, cells were treated with or without hemin for another 2 h and processed for ChIP as described previously ( ) using anti-Bach1 (A1–6), anti-Nrf2 (Santa Cruz Biotechnology, sc-13032), or anti-MafK ( ) antibodies.

Article Title: The bromodomain protein BRD4 regulates the KEAP1/NRF2-dependent oxidative stress response
Article Snippet: The enhancer region E1 was cloned into the BamH1 and SalI restriction sites in the pGL3-basic vector using the primers HMOX1-E1fw: 5′-ACAATTGGCCCAGTCTATGG-3′ and HMOX1-E1rev: 5′-GGAGTTCAAGACCAGCCTGA-3′. .. The following antibodies were used: rabbit polyclonal BRD4 antibody (ab75898, Abcam, Cambridge, UK), mouse monoclonal HMOX1 antibody (ab13243, Abcam), NRF2 (sc13032, sc722, Santa Cruz Biotechnology, Heidelberg, Germany), KEAP1 (K2769, Sigma-Aldrich, Taufkirchen, Germany), alpha-tubulin (T9026, Sigma-Aldrich), and beta-actin (Cell Signaling, Leiden, Netherlands).

Article Title: Hepatitis B virus stimulates G6PD expression through HBx-mediated Nrf2 activation
Article Snippet: Antibodies, reagents, and plasmids The following antibodies were used: anti-β -actin, anti-G6PD (Sigma-Aldrich); anti-HBcAg (Abcam, Cambridge, MA, USA), anti-Keap1 (Cell Signaling, Danvers, MA, USA); anti-Myc, anti-Nrf2 (sc-365949), anti-Lamin B, anti-p62, and anti-Flag (Santa Cruz, Santa Cruz, CA, USA); anti-GFP (BD Biosciences, Franklin Lakes, NJ, USA); goat anti-rabbit IRDye800CW and goat anti-mouse IRDye680 (LI-COR Biosciences, Lincoln, NE, USA). .. For Flag-p62 construction, the p62 coding region was amplified from Cherry-GFP-p62, a gift from Terje Johansen (University of Tromsø, Tromsø, Norway), and was inserted into pCMV-Tag 2A vector using the Eco RI and Xho I restriction sites.

Software:

Article Title: Metabolic Responses in Endothelial Cells Following Exposure to Ketone Bodies
Article Snippet: The following primary antibodies were used: Nrf2 (C-20, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) dilution 1:400 in TBS-T+5% ( w / v ) non-fat dried milk; GAPDH (FL-335, Santa Cruz Biotechnology, Inc.) dilution 1:500 in TBS-T+5% ( w / v ) non-fat dried milk; lamin A/C (N-18, Santa Cruz Biotechnology, Inc.) dilution 1:500 in TBS-T+5% BSA. .. The acquisition of PVDF membrane images and the densitometric analysis of blots was performed using an Alliance MINI HD9 (UVItec) apparatus (Cleaver Scientific, Warwickshire, United Kingdom) and related software.

Real-time Polymerase Chain Reaction:

Article Title: Heme regulates gene expression by triggering Crm1-dependent nuclear export of Bach1
Article Snippet: After 36 h, cells were treated with or without hemin for another 2 h and processed for ChIP as described previously ( ) using anti-Bach1 (A1–6), anti-Nrf2 (Santa Cruz Biotechnology, sc-13032), or anti-MafK ( ) antibodies. .. After 36 h, cells were treated with or without hemin for another 2 h and processed for ChIP as described previously ( ) using anti-Bach1 (A1–6), anti-Nrf2 (Santa Cruz Biotechnology, sc-13032), or anti-MafK ( ) antibodies.

Enzyme-linked Immunosorbent Assay:

Article Title: The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells
Article Snippet: TACS MTT Cell Proliferation Assay Kit and GPX enzyme-linked immunosorbent assay (ELISA) were purchased from R & D Systems (Minneapolis, MN, USA). .. Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Article Title: Sinomenine Regulates Inflammatory Response and Oxidative Stress via Nuclear Factor kappa B (NF-κB) and NF-E2-Related Factor 2 (Nrf2) Signaling Pathways in Ankle Fractures in Children
Article Snippet: We obtained anti-Nrf2 from Santa Cruz Biotechnology (USA), anti-HO-1 was purchased from Enzo Life Sciences (USA), and anti-NQO-1 was obtained from Novus biological (USA). .. IL-1β (cat. no. E-EL-H0149c), IL-6 (cat. no. E-EL-H0102c), and TNF-α (cat. no. E-EL-H0109c) ELISA kits were obtained from Elabscience Biotechnology Co., Ltd. (Wuhan, Hubei, China).

Malondialdehyde MDA Assay:

Article Title: Sinomenine Regulates Inflammatory Response and Oxidative Stress via Nuclear Factor kappa B (NF-κB) and NF-E2-Related Factor 2 (Nrf2) Signaling Pathways in Ankle Fractures in Children
Article Snippet: We obtained anti-Nrf2 from Santa Cruz Biotechnology (USA), anti-HO-1 was purchased from Enzo Life Sciences (USA), and anti-NQO-1 was obtained from Novus biological (USA). .. Malondialdehyde (MDA) assay kits (cat no. ab118970) and superoxide dismutase (SOD) assay kits (cat no. ab65354) were purchased from Abcam (USA).

Fractionation:

Article Title: An Autoregulatory Loop between Nrf2 and Cul3-Rbx1 Controls Their Cellular Abundance *
Article Snippet: The membranes were incubated with anti-Cul3 (1:1000, Cell Signaling), anti-Rbx1 (1:1000, Bio Source) anti-INrf2 (1:1000, Santa Cruz Biotechnology), anti-Nrf2 (1:500, Santa Cruz Biotechnology), anti-FLAG (1:5000, Sigma), anti-V5 (1:5000, Invitrogen), anti-Myc (1:5000, Sigma), or anti-actin (1:5000, Sigma) antibodies, washed, and probed with electrochemiluminescence (Amersham Biosciences). .. To confirm the purity of nuclear-cytoplasmic fractionation, the membranes were reprobed with cytoplasm-specific, anti-lactate dehydrogenase (Chemicon) and nuclear specific, anti-lamin B antibodies (Santa Cruz Biotechnology).

Lysis:

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: Role of NADPH oxidase
Article Snippet: After various treatment, HSCs were washed twice with PBS and scraped in a lysis buffer (20 mM Tris-HCl, 250 mM sucrose, pH 7.2) containing 1X protease inhibitor mixture (Roche, Indianapolis, IN). .. Then, the membranes were probed with the following primary antibodies overnight at 4 °C: anti-α-SMA (1:1000, Abcam, Cambridge, MA), anti-desmin (1:500, BD Pharmingen, San Jose, CA), anti-CB1 (1:1000, Thermo, Waltham, MA), anti-CB2 (1:1000, Abcam), anti-TIMP-1 (1:500, Santa Cruz, Dallas, TX), anti-collagen I (1:1000, Calbiochem, Billerica, MA), anti-Nox1 (1:500, Abcam), anti-Nox4 (1:500, Santa Cruz), anti-Nrf2 (1:1000, Santa Cruz), and anti-β-actin (1:10000, Santa Cruz).

Electrochemiluminescence:

Article Title: An Autoregulatory Loop between Nrf2 and Cul3-Rbx1 Controls Their Cellular Abundance *
Article Snippet: .. The membranes were incubated with anti-Cul3 (1:1000, Cell Signaling), anti-Rbx1 (1:1000, Bio Source) anti-INrf2 (1:1000, Santa Cruz Biotechnology), anti-Nrf2 (1:500, Santa Cruz Biotechnology), anti-FLAG (1:5000, Sigma), anti-V5 (1:5000, Invitrogen), anti-Myc (1:5000, Sigma), or anti-actin (1:5000, Sigma) antibodies, washed, and probed with electrochemiluminescence (Amersham Biosciences). .. To confirm the purity of nuclear-cytoplasmic fractionation, the membranes were reprobed with cytoplasm-specific, anti-lactate dehydrogenase (Chemicon) and nuclear specific, anti-lamin B antibodies (Santa Cruz Biotechnology).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Santa Cruz Biotechnology primary antibodies against nrf2
    Expression of SIRT1, <t>Nrf2,</t> and HO-1 in renal tissues by Western blot analysis ( A ) Western blotting showed protein levels of SIRT1, Nrf2, and HO-1. ( B – D ) Quantitation of Western blot data from (A). Data are presented as the mean ± S.E.M. ( n =6 per group). ★ P
    Primary Antibodies Against Nrf2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/primary antibodies against nrf2/product/Santa Cruz Biotechnology
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    primary antibodies against nrf2 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    99
    Santa Cruz Biotechnology anti nrf2 antibody
    Increased <t>NRF2</t> Activity Confers Chemoresistance BEAS2B cells and cancer cells were exposed to etoposide (A) or carboplatin (B) for 72 h, and viable cells were determined by MTT assay. BEAS2B cells displayed enhanced sensitivity whereas cancer cells with dysfunctional KEAP1 activity demonstrated reduced chemosensitivity to etoposide and carboplatin treatment. Data are presented as percentage of viable cells relative to the vehicle-treated control. Data are the mean of eight independent replicates, combined to generate the mean ± SD for each concentration.
    Anti Nrf2 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti nrf2 antibody/product/Santa Cruz Biotechnology
    Average 99 stars, based on 208 article reviews
    Price from $9.99 to $1999.99
    anti nrf2 antibody - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    75
    Santa Cruz Biotechnology primary antibodies against human nrf2
    Treatment of IMR90 cells with Hexapeptide-11 for 24 h enhances <t>Nrf2</t> nuclear accumulation and upregulates the protein expression levels of proteasomal subunits (A 1 ). Representative immunoblot analyses of Nrf2 expression in cells treated with 2% or 5% (v/v) Hexapeptide-11 (A 2 ). Immunofluorescence images following Nrf2 localization in early passage IMR90 fibroblasts treated with the indicated concentrations of H 2 O 2 or Hexapeptide-11; cells nuclei were counterstained with DAPI. Nrf2 is increasingly distributed in the nucleus (arrows) in H 2 O 2 or Hexapeptide-11 treated cells. (B) Immunoblot analysis of the 20S proteasome subunit α7 expression after cell exposure to the indicated concentrations of Hexapeptide-11. GAPDH probing (A 1 , B) was used as reference for total protein input. Molecular weight markers (in kDa) are indicated on the right of each blot; % (v/v) concentration of Hexapeptide-11 is as in Fig. 1 . Bars, in (A 2 ) 10 µM.
    Primary Antibodies Against Human Nrf2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 75/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/primary antibodies against human nrf2/product/Santa Cruz Biotechnology
    Average 75 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    primary antibodies against human nrf2 - by Bioz Stars, 2020-02
    75/100 stars
      Buy from Supplier

    Image Search Results


    Expression of SIRT1, Nrf2, and HO-1 in renal tissues by Western blot analysis ( A ) Western blotting showed protein levels of SIRT1, Nrf2, and HO-1. ( B – D ) Quantitation of Western blot data from (A). Data are presented as the mean ± S.E.M. ( n =6 per group). ★ P

    Journal: Bioscience Reports

    Article Title: Melatonin attenuates acute kidney ischemia/reperfusion injury in diabetic rats by activation of the SIRT1/Nrf2/HO-1 signaling pathway

    doi: 10.1042/BSR20181614

    Figure Lengend Snippet: Expression of SIRT1, Nrf2, and HO-1 in renal tissues by Western blot analysis ( A ) Western blotting showed protein levels of SIRT1, Nrf2, and HO-1. ( B – D ) Quantitation of Western blot data from (A). Data are presented as the mean ± S.E.M. ( n =6 per group). ★ P

    Article Snippet: Primary antibodies against Nrf2 were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, U.S.A.).

    Techniques: Expressing, Western Blot, Quantitation Assay

    Melatonin pre-treatment up-regulates SIRT1, Nrf2, and HO-1 expression after kidney I/R injury in diabetic rats ( A ) Western blotting showed protein levels of SIRT1, Nrf2, and HO-1. ( B – D ) Quantitation of Western blot data from (A). Data are presented as the mean ± S.E.M. ( n =6 per group). ★ P

    Journal: Bioscience Reports

    Article Title: Melatonin attenuates acute kidney ischemia/reperfusion injury in diabetic rats by activation of the SIRT1/Nrf2/HO-1 signaling pathway

    doi: 10.1042/BSR20181614

    Figure Lengend Snippet: Melatonin pre-treatment up-regulates SIRT1, Nrf2, and HO-1 expression after kidney I/R injury in diabetic rats ( A ) Western blotting showed protein levels of SIRT1, Nrf2, and HO-1. ( B – D ) Quantitation of Western blot data from (A). Data are presented as the mean ± S.E.M. ( n =6 per group). ★ P

    Article Snippet: Primary antibodies against Nrf2 were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, U.S.A.).

    Techniques: Expressing, Western Blot, Quantitation Assay

    Increased NRF2 Activity Confers Chemoresistance BEAS2B cells and cancer cells were exposed to etoposide (A) or carboplatin (B) for 72 h, and viable cells were determined by MTT assay. BEAS2B cells displayed enhanced sensitivity whereas cancer cells with dysfunctional KEAP1 activity demonstrated reduced chemosensitivity to etoposide and carboplatin treatment. Data are presented as percentage of viable cells relative to the vehicle-treated control. Data are the mean of eight independent replicates, combined to generate the mean ± SD for each concentration.

    Journal: PLoS Medicine

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

    doi: 10.1371/journal.pmed.0030420

    Figure Lengend Snippet: Increased NRF2 Activity Confers Chemoresistance BEAS2B cells and cancer cells were exposed to etoposide (A) or carboplatin (B) for 72 h, and viable cells were determined by MTT assay. BEAS2B cells displayed enhanced sensitivity whereas cancer cells with dysfunctional KEAP1 activity demonstrated reduced chemosensitivity to etoposide and carboplatin treatment. Data are presented as percentage of viable cells relative to the vehicle-treated control. Data are the mean of eight independent replicates, combined to generate the mean ± SD for each concentration.

    Article Snippet: Immunohistochemical Staining of NRF2 in NSCLC Tumor Tissue To demonstrate the specificity of anti-NRF2 antibody staining, we preincubated the anti-NRF2 antibody with luciferase (A) and NRF2 (B) in vitro transcribed and translated protein.

    Techniques: Activity Assay, MTT Assay, Concentration Assay

    Mutant KEAP1 Protein Is Unable to Suppress NRF2 Activity (A) Repression activity of the KEAP1 mutants was monitored by a luciferase reporter assay. Wild-type and mutant KEAP1 cDNA constructs were transfected onto H838 cells stably expressing ARE luciferase reporter. Data represent mean ± SD ( n = 3). (B) Silencing of NRF2 by siRNA in A549 cells downregulated the expression of NRF2-dependent genes. A nonspecific siRNA (NS siRNA) was used as control. (C) Inhibition of KEAP1 expression by siRNA in BEAS2B cells upregulated the expression of NRF2-dependent genes.

    Journal: PLoS Medicine

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

    doi: 10.1371/journal.pmed.0030420

    Figure Lengend Snippet: Mutant KEAP1 Protein Is Unable to Suppress NRF2 Activity (A) Repression activity of the KEAP1 mutants was monitored by a luciferase reporter assay. Wild-type and mutant KEAP1 cDNA constructs were transfected onto H838 cells stably expressing ARE luciferase reporter. Data represent mean ± SD ( n = 3). (B) Silencing of NRF2 by siRNA in A549 cells downregulated the expression of NRF2-dependent genes. A nonspecific siRNA (NS siRNA) was used as control. (C) Inhibition of KEAP1 expression by siRNA in BEAS2B cells upregulated the expression of NRF2-dependent genes.

    Article Snippet: Immunohistochemical Staining of NRF2 in NSCLC Tumor Tissue To demonstrate the specificity of anti-NRF2 antibody staining, we preincubated the anti-NRF2 antibody with luciferase (A) and NRF2 (B) in vitro transcribed and translated protein.

    Techniques: Mutagenesis, Activity Assay, Luciferase, Reporter Assay, Construct, Transfection, Stable Transfection, Expressing, Inhibition

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

    Journal: PLoS Medicine

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

    doi: 10.1371/journal.pmed.0030420

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

    Article Snippet: Immunohistochemical Staining of NRF2 in NSCLC Tumor Tissue To demonstrate the specificity of anti-NRF2 antibody staining, we preincubated the anti-NRF2 antibody with luciferase (A) and NRF2 (B) in vitro transcribed and translated protein.

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

    Dysfunctional KEAP1–NRF2 Interaction in NSCLC Tumors (A) Immunohistochemical analysis of NRF2 in NSCLC tissues. Part a shows a patient (PT-18) with mutation in KEAP1 showing strong nuclear and cytoplasmic staining. Part b shows a patient negative for mutation (PT-28) showing weak cytoplasmic staining. Part c shows a patient negative for mutation (PT-20) showing increased nuclear and cytoplasmic staining in tumor tissue. Part d shows weakly staining normal bronchus from the same patient (PT-20). (B) Total GSH and enzyme activities of NQO1 and total GST in NSCLC and matched normal tissues. Raw data for the heat maps are presented in Table S4 . *, samples harboring KEAP1 mutation; §, nmol/mg protein; †, nmol DCPIP reduced/min/mg protein; ‡, nmol of product formed/min/mg protein.

    Journal: PLoS Medicine

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

    doi: 10.1371/journal.pmed.0030420

    Figure Lengend Snippet: Dysfunctional KEAP1–NRF2 Interaction in NSCLC Tumors (A) Immunohistochemical analysis of NRF2 in NSCLC tissues. Part a shows a patient (PT-18) with mutation in KEAP1 showing strong nuclear and cytoplasmic staining. Part b shows a patient negative for mutation (PT-28) showing weak cytoplasmic staining. Part c shows a patient negative for mutation (PT-20) showing increased nuclear and cytoplasmic staining in tumor tissue. Part d shows weakly staining normal bronchus from the same patient (PT-20). (B) Total GSH and enzyme activities of NQO1 and total GST in NSCLC and matched normal tissues. Raw data for the heat maps are presented in Table S4 . *, samples harboring KEAP1 mutation; §, nmol/mg protein; †, nmol DCPIP reduced/min/mg protein; ‡, nmol of product formed/min/mg protein.

    Article Snippet: Immunohistochemical Staining of NRF2 in NSCLC Tumor Tissue To demonstrate the specificity of anti-NRF2 antibody staining, we preincubated the anti-NRF2 antibody with luciferase (A) and NRF2 (B) in vitro transcribed and translated protein.

    Techniques: Immunohistochemistry, Mutagenesis, Staining

    Protective effect of eriodictyol-induced HO-1 expression on H 2 O 2 -induced cell damage. ( A ) H 2 O 2 -exposed cells were pretreated for 1 h with or without ZnPP and then treated with eriodictyol. The inhibitory effect of eriodictyol on H 2 O 2 -induced intracellular ROS generation was observed by fluorescence microscopy; ( B ) H 2 O 2 -stimulated cells were pretreated for 1 h with or without ZnPP or Nrf2 or HO-1 siRNA and then treated with eriodictyol. Protective effect of HO-1 induction on cell death was determined by in situ terminal nick end-labeling (TUNEL) assay: −, untreated; +, treated. ROS and TUNEL staining was quantified in four randomly selected fields for each group: C, control. The scale bars for image ( B ) are the same as that in ( A ), 100 μm. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Eriodictyol Protects Endothelial Cells against Oxidative Stress-Induced Cell Death through Modulating ERK/Nrf2/ARE-Dependent Heme Oxygenase-1 Expression

    doi: 10.3390/ijms160714526

    Figure Lengend Snippet: Protective effect of eriodictyol-induced HO-1 expression on H 2 O 2 -induced cell damage. ( A ) H 2 O 2 -exposed cells were pretreated for 1 h with or without ZnPP and then treated with eriodictyol. The inhibitory effect of eriodictyol on H 2 O 2 -induced intracellular ROS generation was observed by fluorescence microscopy; ( B ) H 2 O 2 -stimulated cells were pretreated for 1 h with or without ZnPP or Nrf2 or HO-1 siRNA and then treated with eriodictyol. Protective effect of HO-1 induction on cell death was determined by in situ terminal nick end-labeling (TUNEL) assay: −, untreated; +, treated. ROS and TUNEL staining was quantified in four randomly selected fields for each group: C, control. The scale bars for image ( B ) are the same as that in ( A ), 100 μm. * p

    Article Snippet: Western Blot Analysis After eriodictyol treatment, cells were washed with phosphate-buffered saline and mixed with RIPA (50 mM Tris CL, pH 7.4/150 mM NaCl/1% Nonidet P-40 (NP-40)/1% sodium deoxycholate/0.1% SDS) buffer containing 1 mM etilendiaminetetraacetic acid (EDTA), 5 µg/mL aprotinin, 2 µg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF), followed by centrifugation at 14,000× g for 15 min. We applied 20 μg of the whole cell lysate protein to each lane and analyzed them by Western blot, using a monoclonal antibody against HO-1, anti-Nrf2, anti-Lamin B, and GAPDH).

    Techniques: Expressing, Fluorescence, Microscopy, In Situ, End Labeling, TUNEL Assay, Staining

    Nrf2 nuclear translocation induced by eriodictyol. ( A ) Cells were treated with 10 and 20 μM eriodictyol at the indicated concentrations for 4 h. Nuclear extracts were subjected to Western blot, using an anti-Nrf2 antibody and anti-lamin B antibody (a marker of nuclear protein); and ( B ) Transient transfection of cells with increasing doses of Nrf2-specific siRNA (10 and 20 nM) reduced HO-1 expression. Western blots representative of three independent experiments are shown: C, untreated cells; +, eriodictyol treatment only; , dose increasing.

    Journal: International Journal of Molecular Sciences

    Article Title: Eriodictyol Protects Endothelial Cells against Oxidative Stress-Induced Cell Death through Modulating ERK/Nrf2/ARE-Dependent Heme Oxygenase-1 Expression

    doi: 10.3390/ijms160714526

    Figure Lengend Snippet: Nrf2 nuclear translocation induced by eriodictyol. ( A ) Cells were treated with 10 and 20 μM eriodictyol at the indicated concentrations for 4 h. Nuclear extracts were subjected to Western blot, using an anti-Nrf2 antibody and anti-lamin B antibody (a marker of nuclear protein); and ( B ) Transient transfection of cells with increasing doses of Nrf2-specific siRNA (10 and 20 nM) reduced HO-1 expression. Western blots representative of three independent experiments are shown: C, untreated cells; +, eriodictyol treatment only; , dose increasing.

    Article Snippet: Western Blot Analysis After eriodictyol treatment, cells were washed with phosphate-buffered saline and mixed with RIPA (50 mM Tris CL, pH 7.4/150 mM NaCl/1% Nonidet P-40 (NP-40)/1% sodium deoxycholate/0.1% SDS) buffer containing 1 mM etilendiaminetetraacetic acid (EDTA), 5 µg/mL aprotinin, 2 µg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF), followed by centrifugation at 14,000× g for 15 min. We applied 20 μg of the whole cell lysate protein to each lane and analyzed them by Western blot, using a monoclonal antibody against HO-1, anti-Nrf2, anti-Lamin B, and GAPDH).

    Techniques: Translocation Assay, Western Blot, Marker, Transfection, Expressing

    Treatment of IMR90 cells with Hexapeptide-11 for 24 h enhances Nrf2 nuclear accumulation and upregulates the protein expression levels of proteasomal subunits (A 1 ). Representative immunoblot analyses of Nrf2 expression in cells treated with 2% or 5% (v/v) Hexapeptide-11 (A 2 ). Immunofluorescence images following Nrf2 localization in early passage IMR90 fibroblasts treated with the indicated concentrations of H 2 O 2 or Hexapeptide-11; cells nuclei were counterstained with DAPI. Nrf2 is increasingly distributed in the nucleus (arrows) in H 2 O 2 or Hexapeptide-11 treated cells. (B) Immunoblot analysis of the 20S proteasome subunit α7 expression after cell exposure to the indicated concentrations of Hexapeptide-11. GAPDH probing (A 1 , B) was used as reference for total protein input. Molecular weight markers (in kDa) are indicated on the right of each blot; % (v/v) concentration of Hexapeptide-11 is as in Fig. 1 . Bars, in (A 2 ) 10 µM.

    Journal: Redox Biology

    Article Title: Hexapeptide-11 is a novel modulator of the proteostasis network in human diploid fibroblasts

    doi: 10.1016/j.redox.2015.04.010

    Figure Lengend Snippet: Treatment of IMR90 cells with Hexapeptide-11 for 24 h enhances Nrf2 nuclear accumulation and upregulates the protein expression levels of proteasomal subunits (A 1 ). Representative immunoblot analyses of Nrf2 expression in cells treated with 2% or 5% (v/v) Hexapeptide-11 (A 2 ). Immunofluorescence images following Nrf2 localization in early passage IMR90 fibroblasts treated with the indicated concentrations of H 2 O 2 or Hexapeptide-11; cells nuclei were counterstained with DAPI. Nrf2 is increasingly distributed in the nucleus (arrows) in H 2 O 2 or Hexapeptide-11 treated cells. (B) Immunoblot analysis of the 20S proteasome subunit α7 expression after cell exposure to the indicated concentrations of Hexapeptide-11. GAPDH probing (A 1 , B) was used as reference for total protein input. Molecular weight markers (in kDa) are indicated on the right of each blot; % (v/v) concentration of Hexapeptide-11 is as in Fig. 1 . Bars, in (A 2 ) 10 µM.

    Article Snippet: Antibodies used Primary antibodies against human Nrf2 (sc-722), proteasome α7 subunit (sc-100456), proteasome β5 subunit (sc-55009), Nqo1 (sc-16464), beclin-1 (sc-11427), p53 (sc-47698), p21CIP1 (sc-6246), and GAPDH (sc-25778), as well as the HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

    Techniques: Expressing, Immunofluorescence, Molecular Weight, Concentration Assay

    Exposure of IMR90 cells to Hexapeptide-11 for 24 h results in the upregulation (mostly dose dependent) of genes involved in the proteostasis network regulation, as well as in cellular antioxidant responses. Shown Q-PCR gene expression analyses refer to 20S and 19S proteasome genes ( α7 , β1 , β2 , β5 , rpn6 , and rpn11 ); to genes involved in ALS functionality ( becn1 , sqstm1 , hdac6 , ctsl and ctsd ) and in molecular chaperones ( hsf1 , hsp27 , hsp70 , hsp90 and clu ), as well as to genes involved in antioxidant responses ( nrf2 , keap1 , nqo1 and txnrd1 ). The beta-2-microglobulin ( b2m ) gene expression was used as normalizer; shown % v/v) concentration of Hexapeptide-11 is as in Fig. 1 . Bars, ±SD. *, P

    Journal: Redox Biology

    Article Title: Hexapeptide-11 is a novel modulator of the proteostasis network in human diploid fibroblasts

    doi: 10.1016/j.redox.2015.04.010

    Figure Lengend Snippet: Exposure of IMR90 cells to Hexapeptide-11 for 24 h results in the upregulation (mostly dose dependent) of genes involved in the proteostasis network regulation, as well as in cellular antioxidant responses. Shown Q-PCR gene expression analyses refer to 20S and 19S proteasome genes ( α7 , β1 , β2 , β5 , rpn6 , and rpn11 ); to genes involved in ALS functionality ( becn1 , sqstm1 , hdac6 , ctsl and ctsd ) and in molecular chaperones ( hsf1 , hsp27 , hsp70 , hsp90 and clu ), as well as to genes involved in antioxidant responses ( nrf2 , keap1 , nqo1 and txnrd1 ). The beta-2-microglobulin ( b2m ) gene expression was used as normalizer; shown % v/v) concentration of Hexapeptide-11 is as in Fig. 1 . Bars, ±SD. *, P

    Article Snippet: Antibodies used Primary antibodies against human Nrf2 (sc-722), proteasome α7 subunit (sc-100456), proteasome β5 subunit (sc-55009), Nqo1 (sc-16464), beclin-1 (sc-11427), p53 (sc-47698), p21CIP1 (sc-6246), and GAPDH (sc-25778), as well as the HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

    Techniques: Polymerase Chain Reaction, Expressing, Concentration Assay

    Prolonged exposure of IMR90 fibroblasts to Hexapeptide-11 upregulates proteasome protein subunits and the antioxidant protein Nqo1 and induces the chymotrypsin-like (LLVY/β5) proteasome peptidase activity in a dose-dependent manner (A 1 ). Immunoblot analyses of the 20S proteasome protein subunits β5 and α7, as well as of Nrf2 and Nqo1 in IMR90 cells treated for 48 h with the indicated concentrations of Hexapeptide-11 (A 2 ). Enzymatic activities of the three (LLVY/β5, LLE/β1 and LRR/β2) proteasome peptidases activities in early passage IMR90 cells treated for 48 and 72 h with 2% or 5% (v/v) Hexapeptide-11 (B 1 ). Relative (%) cathepsin B, L activities in sample preparations from IMR90 exposed to 2% or 5% (v/v) Hexapeptide-11 for 48 and 72 h (B 2 ) immunoblot analyses of the autophagy related protein Beclin-1 in IMR90 cells treated for 48 h with 2% or 5% (v/v) Hexapeptide-11. Proteasome and cathepsin activities were expressed in fluorescence units per µg of input protein vs. controls set to 100%. GAPDH probing in (A 1 , B 2 ) was used as reference for total protein input. Molecular weight markers (in kDa) are indicated on the right of each blot; shown % (v/v) concentration of Hexapeptide-11 is as in Fig. 1 . Bars, ±SD ( n =2). * P

    Journal: Redox Biology

    Article Title: Hexapeptide-11 is a novel modulator of the proteostasis network in human diploid fibroblasts

    doi: 10.1016/j.redox.2015.04.010

    Figure Lengend Snippet: Prolonged exposure of IMR90 fibroblasts to Hexapeptide-11 upregulates proteasome protein subunits and the antioxidant protein Nqo1 and induces the chymotrypsin-like (LLVY/β5) proteasome peptidase activity in a dose-dependent manner (A 1 ). Immunoblot analyses of the 20S proteasome protein subunits β5 and α7, as well as of Nrf2 and Nqo1 in IMR90 cells treated for 48 h with the indicated concentrations of Hexapeptide-11 (A 2 ). Enzymatic activities of the three (LLVY/β5, LLE/β1 and LRR/β2) proteasome peptidases activities in early passage IMR90 cells treated for 48 and 72 h with 2% or 5% (v/v) Hexapeptide-11 (B 1 ). Relative (%) cathepsin B, L activities in sample preparations from IMR90 exposed to 2% or 5% (v/v) Hexapeptide-11 for 48 and 72 h (B 2 ) immunoblot analyses of the autophagy related protein Beclin-1 in IMR90 cells treated for 48 h with 2% or 5% (v/v) Hexapeptide-11. Proteasome and cathepsin activities were expressed in fluorescence units per µg of input protein vs. controls set to 100%. GAPDH probing in (A 1 , B 2 ) was used as reference for total protein input. Molecular weight markers (in kDa) are indicated on the right of each blot; shown % (v/v) concentration of Hexapeptide-11 is as in Fig. 1 . Bars, ±SD ( n =2). * P

    Article Snippet: Antibodies used Primary antibodies against human Nrf2 (sc-722), proteasome α7 subunit (sc-100456), proteasome β5 subunit (sc-55009), Nqo1 (sc-16464), beclin-1 (sc-11427), p53 (sc-47698), p21CIP1 (sc-6246), and GAPDH (sc-25778), as well as the HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

    Techniques: Activity Assay, Fluorescence, Molecular Weight, Concentration Assay