Structured Review

Santa Cruz Biotechnology anti nrf2
Competition between PALB2 and <t>NRF2</t> for KEAP1 binding. (A) Disruption of preformed NRF2/KEAP1 complex in cell extracts. Increasing amounts of whole-cell extracts from 293T cells transfected with pOZC1-PALB2WT or pOZC1-PALB2T92E (lanes 2 and 6, respectively) were added to equal aliquots of extract made from cells cotransfected with HA-NRF2 and Myc-KEAP-GFP. Then, KEAP1 was precipitated using a GFP antibody and the amounts of proteins were analyzed by Western blotting. (B and C) Competitive disruption of isolated NRF2/KEAP1 or PALB2/KEAP1 complexes by IVTed PALB2T551 or NRF2, respectively. See Materials and Methods for detailed procedures. (D) In vitro competition between PALB2 and NRF2 for KEAP1 binding. Different amounts of PALB2 and NRF2 were generated together with a fixed amount of KEAP1 in triple IVT reactions by using different combinations of amounts (in μl) of PCR-generated templates for KEAP1, PALB2, and NRF2 (top panels). Then, KEAP1 was IPed and the amounts of KEAP1, PALB2, and NRF2 in the precipitates were analyzed by Western blotting using anti-HA antibody, which recognizes all 3 proteins (bottom panels).
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1) Product Images from "PALB2 Interacts with KEAP1 To Promote NRF2 Nuclear Accumulation and Function"

Article Title: PALB2 Interacts with KEAP1 To Promote NRF2 Nuclear Accumulation and Function

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.06271-11

Competition between PALB2 and NRF2 for KEAP1 binding. (A) Disruption of preformed NRF2/KEAP1 complex in cell extracts. Increasing amounts of whole-cell extracts from 293T cells transfected with pOZC1-PALB2WT or pOZC1-PALB2T92E (lanes 2 and 6, respectively) were added to equal aliquots of extract made from cells cotransfected with HA-NRF2 and Myc-KEAP-GFP. Then, KEAP1 was precipitated using a GFP antibody and the amounts of proteins were analyzed by Western blotting. (B and C) Competitive disruption of isolated NRF2/KEAP1 or PALB2/KEAP1 complexes by IVTed PALB2T551 or NRF2, respectively. See Materials and Methods for detailed procedures. (D) In vitro competition between PALB2 and NRF2 for KEAP1 binding. Different amounts of PALB2 and NRF2 were generated together with a fixed amount of KEAP1 in triple IVT reactions by using different combinations of amounts (in μl) of PCR-generated templates for KEAP1, PALB2, and NRF2 (top panels). Then, KEAP1 was IPed and the amounts of KEAP1, PALB2, and NRF2 in the precipitates were analyzed by Western blotting using anti-HA antibody, which recognizes all 3 proteins (bottom panels).
Figure Legend Snippet: Competition between PALB2 and NRF2 for KEAP1 binding. (A) Disruption of preformed NRF2/KEAP1 complex in cell extracts. Increasing amounts of whole-cell extracts from 293T cells transfected with pOZC1-PALB2WT or pOZC1-PALB2T92E (lanes 2 and 6, respectively) were added to equal aliquots of extract made from cells cotransfected with HA-NRF2 and Myc-KEAP-GFP. Then, KEAP1 was precipitated using a GFP antibody and the amounts of proteins were analyzed by Western blotting. (B and C) Competitive disruption of isolated NRF2/KEAP1 or PALB2/KEAP1 complexes by IVTed PALB2T551 or NRF2, respectively. See Materials and Methods for detailed procedures. (D) In vitro competition between PALB2 and NRF2 for KEAP1 binding. Different amounts of PALB2 and NRF2 were generated together with a fixed amount of KEAP1 in triple IVT reactions by using different combinations of amounts (in μl) of PCR-generated templates for KEAP1, PALB2, and NRF2 (top panels). Then, KEAP1 was IPed and the amounts of KEAP1, PALB2, and NRF2 in the precipitates were analyzed by Western blotting using anti-HA antibody, which recognizes all 3 proteins (bottom panels).

Techniques Used: Binding Assay, Transfection, Western Blot, Isolation, In Vitro, Generated, Polymerase Chain Reaction

PALB2 binds KEAP1 through a highly conserved ETGE motif. (A) Schematic of PALB2 constructs used in the domain mapping study. (B and C) PALB2 constructs were transiently expressed in 293T cells, proteins were IPed with anti-FLAG M2 agarose, and the precipitates were analyzed by Western blotting. (D) Amino acid sequence alignment of the N-terminal sequences of PALB2 and NRF2 showing the shared LDEETGE KEAP1 binding motif. (E) Deletion or mutations of the ETGE motif in PALB2 all abolish KEAP1 binding. (F) E91R and T92E mutations do not affect the binding of PALB2 to BRCA2, RAD51, and BRCA1.
Figure Legend Snippet: PALB2 binds KEAP1 through a highly conserved ETGE motif. (A) Schematic of PALB2 constructs used in the domain mapping study. (B and C) PALB2 constructs were transiently expressed in 293T cells, proteins were IPed with anti-FLAG M2 agarose, and the precipitates were analyzed by Western blotting. (D) Amino acid sequence alignment of the N-terminal sequences of PALB2 and NRF2 showing the shared LDEETGE KEAP1 binding motif. (E) Deletion or mutations of the ETGE motif in PALB2 all abolish KEAP1 binding. (F) E91R and T92E mutations do not affect the binding of PALB2 to BRCA2, RAD51, and BRCA1.

Techniques Used: Construct, Western Blot, Sequencing, Binding Assay

PALB2 interacts with KEAP1. (A) Domain structure and known binding partners of PALB2. (B) Tandem affinity purification of PALB2 from a HeLa S3 cell line stably expressing a FLAG-HA-double-tagged PALB2 protein (PALB2-FH). Numbers at left are molecular masses in kilodaltons. (C) Reciprocal co-IP of endogenous PALB2 and KEAP1 in U2OS cells. (D) Endogenous KEAP1 association with PALB2 and NRF2 under normal and stress conditions. U2OS cells were treated with DMSO (mock), H 2 O 2 , tBHQ, and CPT at the indicated concentrations for 2 h, and the interactions were analyzed by IP-Western blotting.
Figure Legend Snippet: PALB2 interacts with KEAP1. (A) Domain structure and known binding partners of PALB2. (B) Tandem affinity purification of PALB2 from a HeLa S3 cell line stably expressing a FLAG-HA-double-tagged PALB2 protein (PALB2-FH). Numbers at left are molecular masses in kilodaltons. (C) Reciprocal co-IP of endogenous PALB2 and KEAP1 in U2OS cells. (D) Endogenous KEAP1 association with PALB2 and NRF2 under normal and stress conditions. U2OS cells were treated with DMSO (mock), H 2 O 2 , tBHQ, and CPT at the indicated concentrations for 2 h, and the interactions were analyzed by IP-Western blotting.

Techniques Used: Binding Assay, Affinity Purification, Stable Transfection, Expressing, Co-Immunoprecipitation Assay, Cycling Probe Technology, Western Blot

A proposed model of PALB2 regulation of NRF2 in the nucleus. Nuclear NRF2 forms heterodimers with small Maf proteins and activates antioxidant gene expression through association with the AREs in the promoter region of the genes. KEAP1 represses NRF2 function in the nucleus by blocking NRF2 binding to AREs and exporting it to the cytoplasm for degradation. By competitively binding to KEAP1, PALB2 alleviates the negative impact of KEAP1 on NRF2 function via the two above-noted mechanisms. BRCA2 is also a component of the PALB2-KEAP1 complex, but its role is unclear.
Figure Legend Snippet: A proposed model of PALB2 regulation of NRF2 in the nucleus. Nuclear NRF2 forms heterodimers with small Maf proteins and activates antioxidant gene expression through association with the AREs in the promoter region of the genes. KEAP1 represses NRF2 function in the nucleus by blocking NRF2 binding to AREs and exporting it to the cytoplasm for degradation. By competitively binding to KEAP1, PALB2 alleviates the negative impact of KEAP1 on NRF2 function via the two above-noted mechanisms. BRCA2 is also a component of the PALB2-KEAP1 complex, but its role is unclear.

Techniques Used: Expressing, Blocking Assay, Binding Assay

PALB2 regulates NRF2 nuclear export after induction. (A) Western blotting of whole-cell extracts of U2OS cells (transfected with control or PALB2 siRNAs for 48 h) following treatment with DMSO or tBHQ and at indicated time points after tBHQ removal. (B) Results from 3 independent experiments in panel A were quantified by densitometry and plotted. (C) Western blotting of nuclear extracts of U2OS cells following the same treatments as those in panel A. (D) Results from 3 independent experiments in panel C were quantified by densitometry.
Figure Legend Snippet: PALB2 regulates NRF2 nuclear export after induction. (A) Western blotting of whole-cell extracts of U2OS cells (transfected with control or PALB2 siRNAs for 48 h) following treatment with DMSO or tBHQ and at indicated time points after tBHQ removal. (B) Results from 3 independent experiments in panel A were quantified by densitometry and plotted. (C) Western blotting of nuclear extracts of U2OS cells following the same treatments as those in panel A. (D) Results from 3 independent experiments in panel C were quantified by densitometry.

Techniques Used: Western Blot, Transfection

PALB2 overexpression promotes NRF2 nuclear accumulation and reduces ROS level. (A) An HA-NRF2 plasmid was cotransfected into 293T cells with empty Myc-GFP vector or Myc-PALB2WT-GFP or Myc-PALB2T92E-GFP constructs for 24 h, and their expression and localization were analyzed by fluorescence microscopy. (B) T98G cells overexpressing N-terminally FLAG-HA-double-tagged PALB2 were costained with HA (for PALB2) and NRF2 antibodies and visualized by fluorescence microscopy. (C) T98G cells in panel B were fractionated into cytosol and nuclei, and the amounts of indicated proteins in each compartment were analyzed by Western blotting. (D) Nuclei of HeLa cells harboring the vector or PALB2 overexpression construct were isolated, and protein amounts were analyzed by Western blotting. Lamin A/C, a nuclear protein, was used as a loading control. (E) ROS levels in naïve HeLa cells and the two stable cell lines in panel D were measured using the DCF assay. (F) U2OS cells were cotransfected with the ARE-Luc reporter system and various PALB2 expression vectors, and luciferase activities were measured 48 h after transfection. (G) Cells were transfected with the indicated expression vectors for 30 h, and the tagged PALB2 proteins were IPed with anti-FLAG M2 beads. The IPed PALB2 and co-IPed BRCA2, BRCA1, and KEAP1 proteins were probed with their respective antibodies by Western blotting.
Figure Legend Snippet: PALB2 overexpression promotes NRF2 nuclear accumulation and reduces ROS level. (A) An HA-NRF2 plasmid was cotransfected into 293T cells with empty Myc-GFP vector or Myc-PALB2WT-GFP or Myc-PALB2T92E-GFP constructs for 24 h, and their expression and localization were analyzed by fluorescence microscopy. (B) T98G cells overexpressing N-terminally FLAG-HA-double-tagged PALB2 were costained with HA (for PALB2) and NRF2 antibodies and visualized by fluorescence microscopy. (C) T98G cells in panel B were fractionated into cytosol and nuclei, and the amounts of indicated proteins in each compartment were analyzed by Western blotting. (D) Nuclei of HeLa cells harboring the vector or PALB2 overexpression construct were isolated, and protein amounts were analyzed by Western blotting. Lamin A/C, a nuclear protein, was used as a loading control. (E) ROS levels in naïve HeLa cells and the two stable cell lines in panel D were measured using the DCF assay. (F) U2OS cells were cotransfected with the ARE-Luc reporter system and various PALB2 expression vectors, and luciferase activities were measured 48 h after transfection. (G) Cells were transfected with the indicated expression vectors for 30 h, and the tagged PALB2 proteins were IPed with anti-FLAG M2 beads. The IPed PALB2 and co-IPed BRCA2, BRCA1, and KEAP1 proteins were probed with their respective antibodies by Western blotting.

Techniques Used: Over Expression, Plasmid Preparation, Construct, Expressing, Fluorescence, Microscopy, Western Blot, Isolation, Stable Transfection, DCF Assay, Luciferase, Transfection

siRNA-mediated depletion of PALB2 reduces NRF2 abundance and activity in the nucleus and increases cellular ROS level. (A) Western blotting showing the levels of proteins of interest after siRNA treatments. (B) ROS levels in cells depleted of each of the indicated proteins. (C) Endogenous NRF2 activity on an ARE-luciferase reporter following depletion of PALB2 and other proteins. (D and E) Nuclear NRF2 level after depletion of PALB2 in U2OS cells analyzed by Western blotting (D) and quantified by densitometry (E). (F and G) ChIP analysis of endogenous NRF2 binding to NQO1 ARE in control and PALB2-depleted cells. (F) An agarose gel image of the PCR products in a representative experiment. (G) Results quantified by real-time PCR.
Figure Legend Snippet: siRNA-mediated depletion of PALB2 reduces NRF2 abundance and activity in the nucleus and increases cellular ROS level. (A) Western blotting showing the levels of proteins of interest after siRNA treatments. (B) ROS levels in cells depleted of each of the indicated proteins. (C) Endogenous NRF2 activity on an ARE-luciferase reporter following depletion of PALB2 and other proteins. (D and E) Nuclear NRF2 level after depletion of PALB2 in U2OS cells analyzed by Western blotting (D) and quantified by densitometry (E). (F and G) ChIP analysis of endogenous NRF2 binding to NQO1 ARE in control and PALB2-depleted cells. (F) An agarose gel image of the PCR products in a representative experiment. (G) Results quantified by real-time PCR.

Techniques Used: Activity Assay, Western Blot, Luciferase, Chromatin Immunoprecipitation, Binding Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

2) Product Images from "NRF2 induction supporting breast cancer cell survival is enabled by oxidative stress-induced DPP3-KEAP1 interaction"

Article Title: NRF2 induction supporting breast cancer cell survival is enabled by oxidative stress-induced DPP3-KEAP1 interaction

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-16-2204

DPP3 overexpression promotes NRF2 nuclear accumulation and ROS resistance. ( A ) Levels of DPP3, NRF2 and NQO1 proteins in MCF7 cells stably overexpressing wt or mutant DPP3 proteins. GAPDH was used as a loading control. ( B ) Localization of overexpressed DPP3 and endogenous NRF2 in the MCF7 stable cell lines. Immunofluorescence was carried out using anti-HA and anti-NRF2 antibodies for DPP3 and NRF2, respectively. ( C ) ROS levels in the MCF7 stable cell lines. ( D-E ) Sensitivities of the MCF7 stable cell lines to H 2 O 2 ( D ) and diquat ( E ). Cells were treated with indicated concentrations of H 2 O 2 and diquat for 42 hr. Values presented are means from 2 independent experiments. Error bars represent standard deviations (SDs). Statistical significance was calculated by Student's t test comparing the values for the 2 ETGE mutants (T481E and G482E) with those of 3 ETGE-wt proteins (wt, Y318F and E451Q). *p
Figure Legend Snippet: DPP3 overexpression promotes NRF2 nuclear accumulation and ROS resistance. ( A ) Levels of DPP3, NRF2 and NQO1 proteins in MCF7 cells stably overexpressing wt or mutant DPP3 proteins. GAPDH was used as a loading control. ( B ) Localization of overexpressed DPP3 and endogenous NRF2 in the MCF7 stable cell lines. Immunofluorescence was carried out using anti-HA and anti-NRF2 antibodies for DPP3 and NRF2, respectively. ( C ) ROS levels in the MCF7 stable cell lines. ( D-E ) Sensitivities of the MCF7 stable cell lines to H 2 O 2 ( D ) and diquat ( E ). Cells were treated with indicated concentrations of H 2 O 2 and diquat for 42 hr. Values presented are means from 2 independent experiments. Error bars represent standard deviations (SDs). Statistical significance was calculated by Student's t test comparing the values for the 2 ETGE mutants (T481E and G482E) with those of 3 ETGE-wt proteins (wt, Y318F and E451Q). *p

Techniques Used: Over Expression, Stable Transfection, Mutagenesis, Immunofluorescence

3) Product Images from "NRF2 induction supporting breast cancer cell survival is enabled by oxidative stress-induced DPP3-KEAP1 interaction"

Article Title: NRF2 induction supporting breast cancer cell survival is enabled by oxidative stress-induced DPP3-KEAP1 interaction

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-16-2204

Overexpression of DPP3 enhances the stability of KEAP1. ( A ) Levels of NRF2, DPP3, KEAP1 and p62 in MCF7 cell lines overexpressing wt and mutant DPP3 proteins. β-Actin was used a loading control. ( B-C ) Stabilities of KEAP in the MCF7 cell lines harboring the empty vector or overexpressing wt DPP3. Cells were either untreated or treated with 50 μg/ml of cycloheximide for 2, 4 and 6 hr, and the proteins were analyzed by western blotting. The intensities of KEAP1 bands were quantified by the ImageJ software, normalized against those of GAPDH and plotted. B shows a set of representative western blots, and C shows means of the quantified results from 3 independent experiments. Error bars represent SDs. *p
Figure Legend Snippet: Overexpression of DPP3 enhances the stability of KEAP1. ( A ) Levels of NRF2, DPP3, KEAP1 and p62 in MCF7 cell lines overexpressing wt and mutant DPP3 proteins. β-Actin was used a loading control. ( B-C ) Stabilities of KEAP in the MCF7 cell lines harboring the empty vector or overexpressing wt DPP3. Cells were either untreated or treated with 50 μg/ml of cycloheximide for 2, 4 and 6 hr, and the proteins were analyzed by western blotting. The intensities of KEAP1 bands were quantified by the ImageJ software, normalized against those of GAPDH and plotted. B shows a set of representative western blots, and C shows means of the quantified results from 3 independent experiments. Error bars represent SDs. *p

Techniques Used: Over Expression, Mutagenesis, Plasmid Preparation, Western Blot, Software

4) Product Images from "The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells"

Article Title: The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

Journal: Autophagy

doi: 10.1080/15548627.2015.1061170

NFE2L2 and SQSTM1 are important in the cellular responses to DHA in ARPE-19 cells. ( A ) Cells were transfected with control, NFE2L2 and SQSTM1 siRNA (25 nM) and left for 24 h before reseeding. Following incubation for 24 h, the cells were added DHA (70 μM) or BafA1 (100 nM) for 24 h. Immunoblot for SQSTM1 and MAP1LC3B. ACTB/β-actin and PCNA were used as loading controls. ( B ) The cells were siRNA-transfected as in ( A ). After vehicle (V) and DHA (70 and 140 µM) treatment for 3 h changes in ROS levels were measured using a fluorescent ROS DCF probe. The data are representative for 2 independent experiments both performed in duplicates. The data represent the mean intensity ±SD of 10,000 cells per well and is displayed as relative DCF intensity. ( C ) Relative cell index after transfection with control, NFE2L2 or SQSTM1 siRNA (25 nM) after vehicle and DHA treatment (70 μM) based on real-time monitoring using the xCELLigence instrument. The cell index was normalized to one at the start of the experiment. Mean normalized cell index with standard deviation of triplicate wells of vehicle and DHA treated cells is displayed. Data shown are representative for 3 independent experiments.
Figure Legend Snippet: NFE2L2 and SQSTM1 are important in the cellular responses to DHA in ARPE-19 cells. ( A ) Cells were transfected with control, NFE2L2 and SQSTM1 siRNA (25 nM) and left for 24 h before reseeding. Following incubation for 24 h, the cells were added DHA (70 μM) or BafA1 (100 nM) for 24 h. Immunoblot for SQSTM1 and MAP1LC3B. ACTB/β-actin and PCNA were used as loading controls. ( B ) The cells were siRNA-transfected as in ( A ). After vehicle (V) and DHA (70 and 140 µM) treatment for 3 h changes in ROS levels were measured using a fluorescent ROS DCF probe. The data are representative for 2 independent experiments both performed in duplicates. The data represent the mean intensity ±SD of 10,000 cells per well and is displayed as relative DCF intensity. ( C ) Relative cell index after transfection with control, NFE2L2 or SQSTM1 siRNA (25 nM) after vehicle and DHA treatment (70 μM) based on real-time monitoring using the xCELLigence instrument. The cell index was normalized to one at the start of the experiment. Mean normalized cell index with standard deviation of triplicate wells of vehicle and DHA treated cells is displayed. Data shown are representative for 3 independent experiments.

Techniques Used: Transfection, Incubation, Standard Deviation

The n-3 PUFA DHA increases protein level of SQSTM1 and induces autophagy in ARPE-19 cells. ( A ) Cells were treated with DHA (70 µM) for 24 h and lysed in Triton X-100 (Tx100) buffer. Equal amounts of protein (20 µg) from T × 100 fraction were centrifugated at 10,000 x g and the pellet was dissolved in the same volume of 8 M urea buffer before loading on the gel. The membrane was immunoblotted for SQSTM1 and MAP1LC3B. β-actin (ACTB) and PCNA are used as loading controls. ( B ) The cells were treated with DHA, OA or AA (70 µM) with or without BafA1 (100 nM) for 16 h. Total cell extracts were immunoblotted for SQSTM1. ACTB and PCNA are used as loading controls. ( C ) Protein levels of SQSTM1 and MAP1LC3B determined by immunoblotting of cells treated with DHA (70 µM), BafA1 (100 nM) or a combination of DHA and BafA1 for the indicated time points. The numbers below the MAP1LC3B-II bands represent fold change relative to BafA1 for each time point normalized to PCNA intensity. ACTB and PCNA are used as loading controls. ( D ) The mRNA levels of SQSTM1, MAP1LC3B, MAP1LC3A , and GABARAPL1 relative to ACTB after DHA (70 and 140 µM) supplementation for 16 h determined by quantitative real-time PCR. qRT-PCR data displayed are representative for 2 independent experiments. Mean fold change from triplicate wells ± SD is displayed. Data shown are representative of 3 or more independent experiments, unless otherwise stated.
Figure Legend Snippet: The n-3 PUFA DHA increases protein level of SQSTM1 and induces autophagy in ARPE-19 cells. ( A ) Cells were treated with DHA (70 µM) for 24 h and lysed in Triton X-100 (Tx100) buffer. Equal amounts of protein (20 µg) from T × 100 fraction were centrifugated at 10,000 x g and the pellet was dissolved in the same volume of 8 M urea buffer before loading on the gel. The membrane was immunoblotted for SQSTM1 and MAP1LC3B. β-actin (ACTB) and PCNA are used as loading controls. ( B ) The cells were treated with DHA, OA or AA (70 µM) with or without BafA1 (100 nM) for 16 h. Total cell extracts were immunoblotted for SQSTM1. ACTB and PCNA are used as loading controls. ( C ) Protein levels of SQSTM1 and MAP1LC3B determined by immunoblotting of cells treated with DHA (70 µM), BafA1 (100 nM) or a combination of DHA and BafA1 for the indicated time points. The numbers below the MAP1LC3B-II bands represent fold change relative to BafA1 for each time point normalized to PCNA intensity. ACTB and PCNA are used as loading controls. ( D ) The mRNA levels of SQSTM1, MAP1LC3B, MAP1LC3A , and GABARAPL1 relative to ACTB after DHA (70 and 140 µM) supplementation for 16 h determined by quantitative real-time PCR. qRT-PCR data displayed are representative for 2 independent experiments. Mean fold change from triplicate wells ± SD is displayed. Data shown are representative of 3 or more independent experiments, unless otherwise stated.

Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR

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Clone Assay:

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Centrifugation:

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Luciferase:

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Reporter Assay:

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Synthesized:

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Blocking Assay:

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SYBR Green Assay:

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Incubation:

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Modification:

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Western Blot:

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: Eriodictyol Protects Endothelial Cells against Oxidative Stress-Induced Cell Death through Modulating ERK/Nrf2/ARE-Dependent Heme Oxygenase-1 Expression
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). .. Horseradish peroxidase-conjugated anti-IgG antibodies were used as the secondary antibodies to detect the abovementioned protein bands by enhanced chemiluminescence WESTSAVE-Up ECL kit (AbFrontier, Seoul, Korea).

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: Overexpression of HO-1 Protects against TNF-?-Mediated Airway Inflammation by Down-Regulation of TNFR1-Dependent Oxidative Stress
Article Snippet: Metafectene transfection reagent was from Biontex (Munich, Germany). .. Anti-TNF receptor 1 (TNFR1), Anti-HO-1, anti-Nrf2, anti-ICAM-1, anti-VCAM-1, anti-c-Src, anti-p47phox , and anti-p65 Abs were from Santa Cruz (Santa Cruz, CA).

Article Title: The Role of c-Jun Phosphorylation in EpRE Activation of Phase II Genes
Article Snippet: Fu-GENE 6 transfection reagent was from Roche (Indianapolis, IN). .. Anti-p-c-Jun, anti-Jun, anti Nrf2 and anti-lamin were from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Culture:

Article Title: Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress
Article Snippet: Cell culture medium, DMEM, fetal bovine serum, penicillin–streptomycin cocktail and phosphate buffered saline were purchased from Cellgro Mediatech, Inc. (Herndon, VA). .. Anti-HO-1 (sc-136960), anti-Prx-1 (sc-7381), anti-Trx-1 (sc-166393), anti-Nrf2 (sc-722) and anti-β-actin (sc-47778) were obtained from Santa Cruz (Santa Cruz, CA).

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).

other:

Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway
Article Snippet: Antibodies Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

Article Title: The Crosstalk Between Nrf2 and AMPK Signal Pathways Is Important for the Anti-Inflammatory Effect of Berberine in LPS-Stimulated Macrophages and Endotoxin-Shocked Mice
Article Snippet: Anti-Nrf2, Anti-actin and Anti-H3 antibodies were from Santa Cruz Biotechnologies.

Polymerase Chain Reaction:

Article Title: Mechanism and Significance of Changes in Glutamate-Cysteine Ligase Expression during Hepatic Fibrogenesis *
Article Snippet: The purified DNA was detected by PCR analysis. .. Antibodies used for SeqChIP were anti-Nrf2 and MafG (Santa Cruz Biotechnology).

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: 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).

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).

Isolation:

Article Title: Nrf2-AKT interactions regulate heme oxygenase 1 expression in kidney epithelia during hypoxia and hypoxia-reoxygenation
Article Snippet: Nuclear extracts were isolated using an NI-PER kit (ThermoFisher Scientific). .. Nuclear protein was separated on a 10% SDS-PAGE membrane, and the membrane was probed with anti-Nrf2 or Nuclear Matrix Protein p84 (Nmp-p84) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).

Article Title: The NF-?B Subunit RelA/p65 Is Dispensable for Successful Liver Regeneration after Partial Hepatectomy in Mice
Article Snippet: Paragraph title: Protein Isolation and Immunoblot Analysis ... Antibodies used were: rabbit anti-p65, anti-PCNA, anti-β-actin, anti-Cyclin A, anti-Cyclin D1 (all Santa Cruz), anti-JNK, anti-phospho-JNK, anti-STAT, anti-phospho-STAT (all Cell Signaling).

Purification:

Article Title: Mechanism and Significance of Changes in Glutamate-Cysteine Ligase Expression during Hepatic Fibrogenesis *
Article Snippet: The purified DNA was detected by PCR analysis. .. Antibodies used for SeqChIP were anti-Nrf2 and MafG (Santa Cruz Biotechnology).

Protein Extraction:

Article Title: The Role of c-Jun Phosphorylation in EpRE Activation of Phase II Genes
Article Snippet: Anti-p-c-Jun, anti-Jun, anti Nrf2 and anti-lamin were from Santa Cruz Biotechnology (Santa Cruz, CA). .. M-PER mammalian protein extraction reagent and NE-PER nuclear extraction reagent were from Pierce (Rockford, IL).

Chromatin Immunoprecipitation:

Article Title: Mechanism and Significance of Changes in Glutamate-Cysteine Ligase Expression during Hepatic Fibrogenesis *
Article Snippet: Paragraph title: ChIP and Sequential ChIP (SeqChIP) Assay ... Antibodies used for SeqChIP were anti-Nrf2 and MafG (Santa Cruz Biotechnology).

Article Title: The Role of c-Jun Phosphorylation in EpRE Activation of Phase II Genes
Article Snippet: Anti-p-c-Jun, anti-Jun, anti Nrf2 and anti-lamin were from Santa Cruz Biotechnology (Santa Cruz, CA). .. Chromatin immunoprecipitation (ChIP) assay kit was from Upstate (Chicago, IL).

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: Nrf2-AKT interactions regulate heme oxygenase 1 expression in kidney epithelia during hypoxia and hypoxia-reoxygenation
Article Snippet: .. Nuclear protein was separated on a 10% SDS-PAGE membrane, and the membrane was probed with anti-Nrf2 or Nuclear Matrix Protein p84 (Nmp-p84) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). .. Chromatin immunoprecipitation (ChIP) assays were carried out using a commercially available kit (Upstate Biotechnology, Lake Placid, NY) as detailed elsewhere ( ).

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.

Plasmid Preparation:

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).

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.

Protein Binding:

Article Title: Mechanism and Significance of Changes in Glutamate-Cysteine Ligase Expression during Hepatic Fibrogenesis *
Article Snippet: To examine changes in protein binding to the ARE of the rat GCLC promoter in an endogenous chromatin configuration, ChIP assay was carried out following the ChampionChIPTM kit protocol (SABioscences, Frederick, MD). .. Antibodies used for SeqChIP were anti-Nrf2 and MafG (Santa Cruz Biotechnology).

Immunoprecipitation:

Article Title: Mechanism and Significance of Changes in Glutamate-Cysteine Ligase Expression during Hepatic Fibrogenesis *
Article Snippet: Briefly, DNA immunoprecipitated by Nrf2 antibody was processed for a second round of immunoprecipitation using anti-MafG antibody. .. Antibodies used for SeqChIP were anti-Nrf2 and MafG (Santa Cruz Biotechnology).

Staining:

Article Title: Mechanism and Significance of Changes in Glutamate-Cysteine Ligase Expression during Hepatic Fibrogenesis *
Article Snippet: Antibodies used for SeqChIP were anti-Nrf2 and MafG (Santa Cruz Biotechnology). .. All PCR products were run on 8% acrylamide gels and stained with ethidium bromide for 15–30 min.

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