Structured Review

Santa Cruz Biotechnology anti keap1
Schematic of SILAC-based proteomic mapping of <t>KEAP1</t> modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.
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1) Product Images from "A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling"

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

Journal: Nature

doi: 10.1038/s41586-018-0622-0

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

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

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

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

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

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

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

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

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

Techniques Used:

2) Product Images from "Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress"

Article Title: Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress

Journal: Oncotarget

doi:

Depletion of Keap1 reduces MitoQ-induced autophagy and increases transcriptional activity of the antioxidant Nrf2 (A) MDA-MB-231 cells were treated with increasing concentrations of siRNA oligonucleotides for 24 hr to optimize the downregulation of Keap1. Cells were treated with 10 nM Keap1 siRNA or control NTP siRNA for 24 hr before being treated with MitoQ (1 or 5 μM) for 24 hr. (B) LC3-II protein was used as an autophagy marker. Rapamycin (10 μM) was used as a positive control. (C) Autophagic flux was determined by treating cells with the lysosomal protease inhibitors Pepstatin A (10 μg/ml) and E64d (10 μg/ml) in the presence or absence of MitoQ (1 or 5 μM) for 24 hr. (D) The transcriptional activity of Nrf2 was measured with an assay with immobilized oligonucleotide containing the ARE consensus binding site. tBHQ (20 μM) was used as a positive control. Error bars represent S.D. *statistical significance compared with NTP siRNA cells. (E) Autophagy impairment was measured by observing levels of the autophagy substrate p62.
Figure Legend Snippet: Depletion of Keap1 reduces MitoQ-induced autophagy and increases transcriptional activity of the antioxidant Nrf2 (A) MDA-MB-231 cells were treated with increasing concentrations of siRNA oligonucleotides for 24 hr to optimize the downregulation of Keap1. Cells were treated with 10 nM Keap1 siRNA or control NTP siRNA for 24 hr before being treated with MitoQ (1 or 5 μM) for 24 hr. (B) LC3-II protein was used as an autophagy marker. Rapamycin (10 μM) was used as a positive control. (C) Autophagic flux was determined by treating cells with the lysosomal protease inhibitors Pepstatin A (10 μg/ml) and E64d (10 μg/ml) in the presence or absence of MitoQ (1 or 5 μM) for 24 hr. (D) The transcriptional activity of Nrf2 was measured with an assay with immobilized oligonucleotide containing the ARE consensus binding site. tBHQ (20 μM) was used as a positive control. Error bars represent S.D. *statistical significance compared with NTP siRNA cells. (E) Autophagy impairment was measured by observing levels of the autophagy substrate p62.

Techniques Used: Activity Assay, Multiple Displacement Amplification, Marker, Positive Control, Binding Assay

Depletion of Atg7 inhibits Keap1 degradation in breast cancer cells and MEF (A) MDA-MB-231 cells were transfected with 10 nM Atg7 siRNA or control NTP siRNA for 48 hr before MitoQ treatment. MDA-MB-231 cells were treated with 5 μM of MitoQ, and (B) Atg7 +/+ or Atg7 −/− MEF cells were treated with 5 μM MitoQ. Following 2, 6, or 24 hr of drug exposure, Keap1 degradation was analyzed by Western blot. 50μM tBHQ was used as a positive control. Error bars represent S.D. *statistical significance (p
Figure Legend Snippet: Depletion of Atg7 inhibits Keap1 degradation in breast cancer cells and MEF (A) MDA-MB-231 cells were transfected with 10 nM Atg7 siRNA or control NTP siRNA for 48 hr before MitoQ treatment. MDA-MB-231 cells were treated with 5 μM of MitoQ, and (B) Atg7 +/+ or Atg7 −/− MEF cells were treated with 5 μM MitoQ. Following 2, 6, or 24 hr of drug exposure, Keap1 degradation was analyzed by Western blot. 50μM tBHQ was used as a positive control. Error bars represent S.D. *statistical significance (p

Techniques Used: Multiple Displacement Amplification, Transfection, Western Blot, Positive Control

3) Product Images from "Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis"

Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis

Journal: Scientific Reports

doi: 10.1038/srep44769

Liver expression of Nrf2, Keap1 and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.
Figure Legend Snippet: Liver expression of Nrf2, Keap1 and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.

Techniques Used: Expressing, Immunohistochemistry, Staining

The hepatic expression of Keap1 in liver tissues of patients with PBC and controls. ( A ) Keap1 protein levels were determined with densitometry analyses, after normalization to GAPDH as a loading control. ( B ) Keap1 mRNA levels were estimated in patients with cirrhotic PBC, patients with early stage PBC, and controls. Results were normalized to 18sRNA. Bars indicate the mean ± SEM. ( C ) Representative immunofluorescence micrographs show liver sections from patients with PBC. (a) Nuclei are stained with DAPI (blue). (b) Immunofluorescence staining of Keap1 (green) shows its abundance in hepatocytes. (c) Arrows indicate the perinuclear and nuclear localizations of Keap1whereas arrowheads indicate cytoplasmic localization of Keap1.
Figure Legend Snippet: The hepatic expression of Keap1 in liver tissues of patients with PBC and controls. ( A ) Keap1 protein levels were determined with densitometry analyses, after normalization to GAPDH as a loading control. ( B ) Keap1 mRNA levels were estimated in patients with cirrhotic PBC, patients with early stage PBC, and controls. Results were normalized to 18sRNA. Bars indicate the mean ± SEM. ( C ) Representative immunofluorescence micrographs show liver sections from patients with PBC. (a) Nuclei are stained with DAPI (blue). (b) Immunofluorescence staining of Keap1 (green) shows its abundance in hepatocytes. (c) Arrows indicate the perinuclear and nuclear localizations of Keap1whereas arrowheads indicate cytoplasmic localization of Keap1.

Techniques Used: Expressing, Immunofluorescence, Staining

4) Product Images from "Time-dependent effects of systemic lipopolysaccharide injection on regulators of antioxidant defense Nrf2 and PGC-1 ? in neonatal rat brain."

Article Title: Time-dependent effects of systemic lipopolysaccharide injection on regulators of antioxidant defense Nrf2 and PGC-1 ? in neonatal rat brain.

Journal: Neuroimmunomodulation

doi: 10.1159/000347161

Systemic LPS (0.3 mg/kg i.p.) induced the antioxidant defence systems after 24 h. Seven-day old rat pups were exposed to either saline (n = 9) or LPS (0.3 mg/kg) (n = 11) for 24 h after which their brains were removed for the protein expression analysis of Nrf2, Keap1, γGCL-C and γGCL-M subunits was analysed (A). In (B), the densitometric analysis of western blot is shown. Statistics: *p
Figure Legend Snippet: Systemic LPS (0.3 mg/kg i.p.) induced the antioxidant defence systems after 24 h. Seven-day old rat pups were exposed to either saline (n = 9) or LPS (0.3 mg/kg) (n = 11) for 24 h after which their brains were removed for the protein expression analysis of Nrf2, Keap1, γGCL-C and γGCL-M subunits was analysed (A). In (B), the densitometric analysis of western blot is shown. Statistics: *p

Techniques Used: Expressing, Western Blot

Systemic LPS (0.3 mg/kg i.p.) caused decreased antioxidant defence levels after 72 h. (A) Seven-day old rat pups were injected with saline (n = 11) or LPS (0.3 mg/kg) (n = 9) and after 72 h their brains were removed for protein level analysis of Nrf2, Keap1, γGCL-C and γGCL-M subunits. The densitometric analysis of western blot is shown in (B). Statistics: *p
Figure Legend Snippet: Systemic LPS (0.3 mg/kg i.p.) caused decreased antioxidant defence levels after 72 h. (A) Seven-day old rat pups were injected with saline (n = 11) or LPS (0.3 mg/kg) (n = 9) and after 72 h their brains were removed for protein level analysis of Nrf2, Keap1, γGCL-C and γGCL-M subunits. The densitometric analysis of western blot is shown in (B). Statistics: *p

Techniques Used: Injection, Western Blot

5) 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

6) Product Images from "Isodeoxyelephantopin induces protective autophagy in lung cancer cells via Nrf2-p62-keap1 feedback loop"

Article Title: Isodeoxyelephantopin induces protective autophagy in lung cancer cells via Nrf2-p62-keap1 feedback loop

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.265

p62 is required for ESI-induced autophagy. ( a ) H1299 and A549 cells were treated with the indicated concentration of ESI for 24 h, and the p62 protein level was determined by western blot. ( b ) H1299 cells transfected with Flag-Keap1 plasmid were treated with ESI (1.6 μ M) for 24 h. Immunoprecipitation was performed using an anti-Flag antibody or IgG as control, and immunublotting was carried out on the total cell lysates or immunoprecipitates using the indicated antibodies. ( c ) H1299 and A549 cells were transfected with pEGFP-p62 plasmid and pEGFP-N1 (vector control), and the autophagy markers including Beclin1, ATG3 and LC3 were determined by western blot. ( d - f ) H1299 cells overexpressing mCherry-LC3 were transfected with pEGFP-p62 plasmid and pEGFP-N1 (vector control) for 24 h, and the mCherry-LC3 punctate dots in cells were examined. Positive signals were defined if the cells have five or more mCherry-LC3 dots in the cytoplasm. ( e and f ) The number of mCherry-LC3 dots per cell ( e ) and the percentage of the cells with mCherry-LC3 dots ( f ) were counted under florescence microscope. ( g and h ) H1299 and A549 cells were transfected with anti-p62 siRNA (50 nM) or control siRNA together with indicated concentrations of ESI treatment, and expression levels of the autophagy markers ( g ) and cell viability ( h ) were analyzed. Note that knockdown of p62 attenuated ESI-induced autophagy and survival. All data were representative of three independent experiments. Bars, S.E.M.; * P
Figure Legend Snippet: p62 is required for ESI-induced autophagy. ( a ) H1299 and A549 cells were treated with the indicated concentration of ESI for 24 h, and the p62 protein level was determined by western blot. ( b ) H1299 cells transfected with Flag-Keap1 plasmid were treated with ESI (1.6 μ M) for 24 h. Immunoprecipitation was performed using an anti-Flag antibody or IgG as control, and immunublotting was carried out on the total cell lysates or immunoprecipitates using the indicated antibodies. ( c ) H1299 and A549 cells were transfected with pEGFP-p62 plasmid and pEGFP-N1 (vector control), and the autophagy markers including Beclin1, ATG3 and LC3 were determined by western blot. ( d - f ) H1299 cells overexpressing mCherry-LC3 were transfected with pEGFP-p62 plasmid and pEGFP-N1 (vector control) for 24 h, and the mCherry-LC3 punctate dots in cells were examined. Positive signals were defined if the cells have five or more mCherry-LC3 dots in the cytoplasm. ( e and f ) The number of mCherry-LC3 dots per cell ( e ) and the percentage of the cells with mCherry-LC3 dots ( f ) were counted under florescence microscope. ( g and h ) H1299 and A549 cells were transfected with anti-p62 siRNA (50 nM) or control siRNA together with indicated concentrations of ESI treatment, and expression levels of the autophagy markers ( g ) and cell viability ( h ) were analyzed. Note that knockdown of p62 attenuated ESI-induced autophagy and survival. All data were representative of three independent experiments. Bars, S.E.M.; * P

Techniques Used: Concentration Assay, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Microscopy, Expressing

Schematic diagram summarizing the ESI-induced Nrf2-p62-Keap1 feedback loop and protective autophagy
Figure Legend Snippet: Schematic diagram summarizing the ESI-induced Nrf2-p62-Keap1 feedback loop and protective autophagy

Techniques Used:

7) Product Images from "Brd4 regulates the expression of essential autophagy genes and Keap1 in AML cells"

Article Title: Brd4 regulates the expression of essential autophagy genes and Keap1 in AML cells

Journal: Oncotarget

doi: 10.18632/oncotarget.24432

Effects of Keap1 depletion on p62 and LC3B; effects of Keap1 or Nrf2 depletion on Nrf2 NQO1, and apoptosis induced by Brd4 inhibition (A) Effect of Keap1 depletion by CRISPR-cas9 mediated genome editing on p62, LC3B, Nrf2, GCLC, GCLM, and NQO1 in OCI-AML3 cells. OCI-AML3 cells stably expressing inducible KEAP1 gRNA were treated with vehicle or doxycycline to induce genome editing of KEAP1 for six days, followed by Western blotting of the proteins shown. (B) Effects of CRISPR-cas9 mediated genome editing of KEAP1 on JQ1-induced apoptosis. OCI-AML3 cells stably expressing inducible KEAP1 gRNA were incubated with or without doxycycline for five days, followed by treatment with JQ1 at the indicated concentrations for 48 h and analysis of Annexin V positivity. Bar graphs represent the mean ± S.D. of biological triplicates. Asterisks ( ** ) and ( *** ) indicate p
Figure Legend Snippet: Effects of Keap1 depletion on p62 and LC3B; effects of Keap1 or Nrf2 depletion on Nrf2 NQO1, and apoptosis induced by Brd4 inhibition (A) Effect of Keap1 depletion by CRISPR-cas9 mediated genome editing on p62, LC3B, Nrf2, GCLC, GCLM, and NQO1 in OCI-AML3 cells. OCI-AML3 cells stably expressing inducible KEAP1 gRNA were treated with vehicle or doxycycline to induce genome editing of KEAP1 for six days, followed by Western blotting of the proteins shown. (B) Effects of CRISPR-cas9 mediated genome editing of KEAP1 on JQ1-induced apoptosis. OCI-AML3 cells stably expressing inducible KEAP1 gRNA were incubated with or without doxycycline for five days, followed by treatment with JQ1 at the indicated concentrations for 48 h and analysis of Annexin V positivity. Bar graphs represent the mean ± S.D. of biological triplicates. Asterisks ( ** ) and ( *** ) indicate p

Techniques Used: Inhibition, CRISPR, Stable Transfection, Expressing, Western Blot, Incubation

Effects of JQ1 on the Nrf2 antioxidant pathway (A) OCI-AML3 cells were cultured in the presence or absence of 500 nM JQ1 for 24 h, followed by Brd4- and CEBPβ-ChIP-seq. The representative genome browser views of Brd4- or CEBPβ-binding peaks adjacent to the Keap1 locus are shown. (B) OCI-AML3 cells were treated with indicated concentrations of JQ1 for 24 h and 48 h, followed by q-PCR analysis of Keap1 mRNA expression. (C, D, E, F, G, H) OCI-AML3 cells were treated with JQ1 or I-BET-151 at the indicated concentrations for 24 h, followed by Western blotting (C, F), qPCR analysis of Nrf2, GCLC, GCLM, and NQO1 (D, G), and quantitative measurement of superoxide and hydrogen peroxide production (E, H).
Figure Legend Snippet: Effects of JQ1 on the Nrf2 antioxidant pathway (A) OCI-AML3 cells were cultured in the presence or absence of 500 nM JQ1 for 24 h, followed by Brd4- and CEBPβ-ChIP-seq. The representative genome browser views of Brd4- or CEBPβ-binding peaks adjacent to the Keap1 locus are shown. (B) OCI-AML3 cells were treated with indicated concentrations of JQ1 for 24 h and 48 h, followed by q-PCR analysis of Keap1 mRNA expression. (C, D, E, F, G, H) OCI-AML3 cells were treated with JQ1 or I-BET-151 at the indicated concentrations for 24 h, followed by Western blotting (C, F), qPCR analysis of Nrf2, GCLC, GCLM, and NQO1 (D, G), and quantitative measurement of superoxide and hydrogen peroxide production (E, H).

Techniques Used: Cell Culture, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Expressing, Western Blot, Real-time Polymerase Chain Reaction

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

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-26049-5

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

Techniques Used: Expressing, Western Blot

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

Techniques Used: Methylation, Clone Assay, Sequencing

9) Product Images from "Cytotoxic Effect of Thymoquinone-Loaded Nanostructured Lipid Carrier (TQ-NLC) on Liver Cancer Cell Integrated with Hepatitis B Genome, Hep3B"

Article Title: Cytotoxic Effect of Thymoquinone-Loaded Nanostructured Lipid Carrier (TQ-NLC) on Liver Cancer Cell Integrated with Hepatitis B Genome, Hep3B

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2018/1549805

Keap1 expression in Hep3B upon treatment with TQ or TQ-NLC. (a) Western blot analysis of Keap1 protein expression after treatment with indicated concentrations of TQ or TQ-NLC for 12, 24, 48, and 72 hours. Whole cell protein lysates were analyzed via Western blotting using antibodies against Keap1. (b) Protein levels were quantified using the densitometry analysis of Image J and expressed as a percentage of relative density. Data are presented as mean ± SEM and represent three independent experiments. Statistically significant differences are indicated as ( ∗ p
Figure Legend Snippet: Keap1 expression in Hep3B upon treatment with TQ or TQ-NLC. (a) Western blot analysis of Keap1 protein expression after treatment with indicated concentrations of TQ or TQ-NLC for 12, 24, 48, and 72 hours. Whole cell protein lysates were analyzed via Western blotting using antibodies against Keap1. (b) Protein levels were quantified using the densitometry analysis of Image J and expressed as a percentage of relative density. Data are presented as mean ± SEM and represent three independent experiments. Statistically significant differences are indicated as ( ∗ p

Techniques Used: Expressing, Western Blot

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

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

Journal: Toxicology Reports

doi: 10.1016/j.toxrep.2014.11.015

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

Techniques Used: Western Blot, Polyacrylamide Gel Electrophoresis

11) Product Images from "Nestin regulates cellular redox homeostasis in lung cancer through the Keap1–Nrf2 feedback loop"

Article Title: Nestin regulates cellular redox homeostasis in lung cancer through the Keap1–Nrf2 feedback loop

Journal: Nature Communications

doi: 10.1038/s41467-019-12925-9

The ESGE motif is essential for the ability of Nestin to interact with Keap1. a Schematic depiction of wild-type and deletion mutants of Myc-tagged Nestin and Flag-tagged Keap1. b A series of truncated Myc-tagged Nestin proteins were expressed with Flag-tagged Keap1 in HEK293FT cells. Immunoprecipitation was performed using Protein G beads and an anti-Flag antibody. c Truncated Flag-tagged Keap1 proteins were expressed with Myc-tagged Nestin in HEK293FT cells. d Nestin-knockdown NSCLC cells were transfected with empty pcDNA3.1, the same vector encoding Myc-Nestin or Nestin (N1295-1621). At 48 h post-transfection, the cells were transfected with the ARE luciferase reporter and subsequently assayed for luciferase activity. e Comparison of the mRNA expression levels of Nrf2-downstream genes in Nestin-knockdown NSCLC cells transfected with or without pcDNA3.1, Myc-Nestin or Nestin (N1295-1621) vectors. f Nestin-knockdown NSCLC cells were, respectively, transfected with pcDNA3.1, Myc-Nestin, or Nestin (N1295-1621) as indicated. The expression levels of Nrf2, NQO1, and HO-1 were analyzed via immunoblotting. g The total antioxidant activity (T-AOC) of the NSCLC cells described in f was assessed. h Nestin-knockdown NSCLC cells were transfected with pcDNA3.1, Myc-Nestin, or truncated Nestin (N1295-1621) plasmids for 72 h and then treated with 200 μM H 2 O 2 for 4 h. Flow cytometry with Annexin V-FITC and PI was used to detect apoptosis. i Statistical analysis of the total apoptosis rates in the NSCLC cells described in h . j Highlighted sequence alignment of the putative Keap1-binding motif in Nestin from different species and those previously reported in Nrf2 and PGAM5. k NSCLC cells were transfected with plasmids encoding Myc-Nestin, ΔESGE, or ESGA mutants. Immunoprecipitation was performed using Protein G beads and an anti-Myc antibody. l NSCLC cells were transfected with plasmids encoding Myc-Nestin, ΔESGE, or ESGA mutants. Lysates were denatured, immunoprecipitated with anti-Nrf2, and blotted with anti-ubiquitin. Data are presented as the means ± SD of three independent experiments. * P
Figure Legend Snippet: The ESGE motif is essential for the ability of Nestin to interact with Keap1. a Schematic depiction of wild-type and deletion mutants of Myc-tagged Nestin and Flag-tagged Keap1. b A series of truncated Myc-tagged Nestin proteins were expressed with Flag-tagged Keap1 in HEK293FT cells. Immunoprecipitation was performed using Protein G beads and an anti-Flag antibody. c Truncated Flag-tagged Keap1 proteins were expressed with Myc-tagged Nestin in HEK293FT cells. d Nestin-knockdown NSCLC cells were transfected with empty pcDNA3.1, the same vector encoding Myc-Nestin or Nestin (N1295-1621). At 48 h post-transfection, the cells were transfected with the ARE luciferase reporter and subsequently assayed for luciferase activity. e Comparison of the mRNA expression levels of Nrf2-downstream genes in Nestin-knockdown NSCLC cells transfected with or without pcDNA3.1, Myc-Nestin or Nestin (N1295-1621) vectors. f Nestin-knockdown NSCLC cells were, respectively, transfected with pcDNA3.1, Myc-Nestin, or Nestin (N1295-1621) as indicated. The expression levels of Nrf2, NQO1, and HO-1 were analyzed via immunoblotting. g The total antioxidant activity (T-AOC) of the NSCLC cells described in f was assessed. h Nestin-knockdown NSCLC cells were transfected with pcDNA3.1, Myc-Nestin, or truncated Nestin (N1295-1621) plasmids for 72 h and then treated with 200 μM H 2 O 2 for 4 h. Flow cytometry with Annexin V-FITC and PI was used to detect apoptosis. i Statistical analysis of the total apoptosis rates in the NSCLC cells described in h . j Highlighted sequence alignment of the putative Keap1-binding motif in Nestin from different species and those previously reported in Nrf2 and PGAM5. k NSCLC cells were transfected with plasmids encoding Myc-Nestin, ΔESGE, or ESGA mutants. Immunoprecipitation was performed using Protein G beads and an anti-Myc antibody. l NSCLC cells were transfected with plasmids encoding Myc-Nestin, ΔESGE, or ESGA mutants. Lysates were denatured, immunoprecipitated with anti-Nrf2, and blotted with anti-ubiquitin. Data are presented as the means ± SD of three independent experiments. * P

Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Antioxidant Activity Assay, Flow Cytometry, Cytometry, Sequencing, Binding Assay

Nestin interferes with the Keap1-dependent ubiquitination of Nrf2 by competitively binding to Keap1. a qPCR analysis showing that knockdown of Nestin had no effect on Keap1 expression at the mRNA level. b Immunoblotting analysis showing that Nestin had no effect on Keap1 expression at the protein level. c Alteration of the Nestin levels had no influence on the ubiquitination of Keap1. Control or Nestin-knockdown cells transfected with or without a vector encoding Myc-Nestin were treated with 10 μM MG132 for 4 h and an in vivo ubiquitination assay was performed to determine the ubiquitination levels of Keap1. d Myc-Nestin plasmids were transfected into NSCLC cells, whole-cell lysates were immunoprecipitated with anti-Myc, and the precipitated proteins were blotted with the indicated antibodies. e Whole-cell lysates were immunoprecipitated with anti-Keap1 and the precipitated proteins were blotted with anti-Nestin, anti-Keap1, and anti-Nrf2. f The localizations of endogenous Keap1 and Nestin in NSCLC cells were determined by double-label indirect immunofluorescence with anti-Keap1 (red) and anti-Nestin (green) antibodies. The colocalization of Keap1 and Nestin is indicated by a yellow color in the merged images. Scale bar: 5 μm. g Nestin reduced the interaction between Nrf2 and Keap1. Control or Nestin-knockdown NSCLC cells were treated with 10 μM MG132 for 4 h. Cell lysates were immunoprecipitated with an anti-Keap1 antibody and blotted with an anti-Nrf2 antibody. N.S. represents no significant, Student’s t test. Source data are available as a Source Data file
Figure Legend Snippet: Nestin interferes with the Keap1-dependent ubiquitination of Nrf2 by competitively binding to Keap1. a qPCR analysis showing that knockdown of Nestin had no effect on Keap1 expression at the mRNA level. b Immunoblotting analysis showing that Nestin had no effect on Keap1 expression at the protein level. c Alteration of the Nestin levels had no influence on the ubiquitination of Keap1. Control or Nestin-knockdown cells transfected with or without a vector encoding Myc-Nestin were treated with 10 μM MG132 for 4 h and an in vivo ubiquitination assay was performed to determine the ubiquitination levels of Keap1. d Myc-Nestin plasmids were transfected into NSCLC cells, whole-cell lysates were immunoprecipitated with anti-Myc, and the precipitated proteins were blotted with the indicated antibodies. e Whole-cell lysates were immunoprecipitated with anti-Keap1 and the precipitated proteins were blotted with anti-Nestin, anti-Keap1, and anti-Nrf2. f The localizations of endogenous Keap1 and Nestin in NSCLC cells were determined by double-label indirect immunofluorescence with anti-Keap1 (red) and anti-Nestin (green) antibodies. The colocalization of Keap1 and Nestin is indicated by a yellow color in the merged images. Scale bar: 5 μm. g Nestin reduced the interaction between Nrf2 and Keap1. Control or Nestin-knockdown NSCLC cells were treated with 10 μM MG132 for 4 h. Cell lysates were immunoprecipitated with an anti-Keap1 antibody and blotted with an anti-Nrf2 antibody. N.S. represents no significant, Student’s t test. Source data are available as a Source Data file

Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation, In Vivo, Ubiquitin Assay, Immunoprecipitation, Immunofluorescence

12) Product Images from "Protective action of glutamine in rats with severe acute liver failure"

Article Title: Protective action of glutamine in rats with severe acute liver failure

Journal: World Journal of Hepatology

doi: 10.4254/wjh.v11.i3.273

Effect of glutamine in experimental severe acute liver failure. Western blot analysis of protein expression of (A) Nrf2 and (B) Keap1. Values expressed as mean ± standard error. b P
Figure Legend Snippet: Effect of glutamine in experimental severe acute liver failure. Western blot analysis of protein expression of (A) Nrf2 and (B) Keap1. Values expressed as mean ± standard error. b P

Techniques Used: Western Blot, Expressing

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

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

Journal: Breast cancer research and treatment

doi: 10.1007/s10549-011-1604-1

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

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

14) Product Images from "Cytotoxic Effect of Thymoquinone-Loaded Nanostructured Lipid Carrier (TQ-NLC) on Liver Cancer Cell Integrated with Hepatitis B Genome, Hep3B"

Article Title: Cytotoxic Effect of Thymoquinone-Loaded Nanostructured Lipid Carrier (TQ-NLC) on Liver Cancer Cell Integrated with Hepatitis B Genome, Hep3B

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2018/1549805

Keap1 expression in Hep3B upon treatment with TQ or TQ-NLC. (a) Western blot analysis of Keap1 protein expression after treatment with indicated concentrations of TQ or TQ-NLC for 12, 24, 48, and 72 hours. Whole cell protein lysates were analyzed via Western blotting using antibodies against Keap1. (b) Protein levels were quantified using the densitometry analysis of Image J and expressed as a percentage of relative density. Data are presented as mean ± SEM and represent three independent experiments. Statistically significant differences are indicated as ( ∗ p
Figure Legend Snippet: Keap1 expression in Hep3B upon treatment with TQ or TQ-NLC. (a) Western blot analysis of Keap1 protein expression after treatment with indicated concentrations of TQ or TQ-NLC for 12, 24, 48, and 72 hours. Whole cell protein lysates were analyzed via Western blotting using antibodies against Keap1. (b) Protein levels were quantified using the densitometry analysis of Image J and expressed as a percentage of relative density. Data are presented as mean ± SEM and represent three independent experiments. Statistically significant differences are indicated as ( ∗ p

Techniques Used: Expressing, Western Blot

15) Product Images from "Brd4 regulates the expression of essential autophagy genes and Keap1 in AML cells"

Article Title: Brd4 regulates the expression of essential autophagy genes and Keap1 in AML cells

Journal: Oncotarget

doi: 10.18632/oncotarget.24432

Effects of Keap1 depletion on p62 and LC3B; effects of Keap1 or Nrf2 depletion on Nrf2 NQO1, and apoptosis induced by Brd4 inhibition (A) Effect of Keap1 depletion by CRISPR-cas9 mediated genome editing on p62, LC3B, Nrf2, GCLC, GCLM, and NQO1 in OCI-AML3 cells. OCI-AML3 cells stably expressing inducible KEAP1 gRNA were treated with vehicle or doxycycline to induce genome editing of KEAP1 for six days, followed by Western blotting of the proteins shown. (B) Effects of CRISPR-cas9 mediated genome editing of KEAP1 on JQ1-induced apoptosis. OCI-AML3 cells stably expressing inducible KEAP1 gRNA were incubated with or without doxycycline for five days, followed by treatment with JQ1 at the indicated concentrations for 48 h and analysis of Annexin V positivity. Bar graphs represent the mean ± S.D. of biological triplicates. Asterisks ( ** ) and ( *** ) indicate p
Figure Legend Snippet: Effects of Keap1 depletion on p62 and LC3B; effects of Keap1 or Nrf2 depletion on Nrf2 NQO1, and apoptosis induced by Brd4 inhibition (A) Effect of Keap1 depletion by CRISPR-cas9 mediated genome editing on p62, LC3B, Nrf2, GCLC, GCLM, and NQO1 in OCI-AML3 cells. OCI-AML3 cells stably expressing inducible KEAP1 gRNA were treated with vehicle or doxycycline to induce genome editing of KEAP1 for six days, followed by Western blotting of the proteins shown. (B) Effects of CRISPR-cas9 mediated genome editing of KEAP1 on JQ1-induced apoptosis. OCI-AML3 cells stably expressing inducible KEAP1 gRNA were incubated with or without doxycycline for five days, followed by treatment with JQ1 at the indicated concentrations for 48 h and analysis of Annexin V positivity. Bar graphs represent the mean ± S.D. of biological triplicates. Asterisks ( ** ) and ( *** ) indicate p

Techniques Used: Inhibition, CRISPR, Stable Transfection, Expressing, Western Blot, Incubation

Effects of JQ1 on the Nrf2 antioxidant pathway (A) OCI-AML3 cells were cultured in the presence or absence of 500 nM JQ1 for 24 h, followed by Brd4- and CEBPβ-ChIP-seq. The representative genome browser views of Brd4- or CEBPβ-binding peaks adjacent to the Keap1 locus are shown. (B) OCI-AML3 cells were treated with indicated concentrations of JQ1 for 24 h and 48 h, followed by q-PCR analysis of Keap1 mRNA expression. (C, D, E, F, G, H) OCI-AML3 cells were treated with JQ1 or I-BET-151 at the indicated concentrations for 24 h, followed by Western blotting (C, F), qPCR analysis of Nrf2, GCLC, GCLM, and NQO1 (D, G), and quantitative measurement of superoxide and hydrogen peroxide production (E, H).
Figure Legend Snippet: Effects of JQ1 on the Nrf2 antioxidant pathway (A) OCI-AML3 cells were cultured in the presence or absence of 500 nM JQ1 for 24 h, followed by Brd4- and CEBPβ-ChIP-seq. The representative genome browser views of Brd4- or CEBPβ-binding peaks adjacent to the Keap1 locus are shown. (B) OCI-AML3 cells were treated with indicated concentrations of JQ1 for 24 h and 48 h, followed by q-PCR analysis of Keap1 mRNA expression. (C, D, E, F, G, H) OCI-AML3 cells were treated with JQ1 or I-BET-151 at the indicated concentrations for 24 h, followed by Western blotting (C, F), qPCR analysis of Nrf2, GCLC, GCLM, and NQO1 (D, G), and quantitative measurement of superoxide and hydrogen peroxide production (E, H).

Techniques Used: Cell Culture, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Expressing, Western Blot, Real-time Polymerase Chain Reaction

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

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

Journal: Molecules (Basel, Switzerland)

doi: 10.3390/molecules15053338

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

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

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

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

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.275495

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

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

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

Techniques Used: Expressing, Multiple Displacement Amplification

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

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

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

Techniques Used: Activity Assay, Expressing

18) 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:

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:

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

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-27123-8

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

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

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

Techniques Used: Expressing

20) Product Images from "Gankyrin has an antioxidative role through the feedback regulation of Nrf2 in hepatocellular carcinoma"

Article Title: Gankyrin has an antioxidative role through the feedback regulation of Nrf2 in hepatocellular carcinoma

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20151208

Gankyrin binds to the Kelch domain of Keap1. (A) Gankyrin influenced the binding of Keap1 to Nrf2. Equal amounts of cell lysates were immunoprecipitated with an anti-Keap1 antibody. Precipitated proteins and cell lysates were blotted with anti-Nrf2, anti-gankyrin, and anti-Keap1 antibodies. (B) Confocal microscopy was performed on HEK293T cells cotransfected with Keap1 and myc-gankyrin. Bar, 10 µm. (C and D) Gankyrin and Keap1 were cotransfected into 293T cells. Whole cell lysates were immunoprecipitated with Keap1- (C) or gankyrin-specific (D) antibodies. Precipitated proteins and cell lysates were blotted with the indicated antibodies. (E) Cell lysates from SMMC7721-con and SMMC7721-ovGank cells were immunoprecipitated with anti-Keap1 antibodies, and Western blot analysis was performed with the indicated antibodies. (F) The interaction of Myc-gankyrin with Flag-tagged truncated Keap1 fragments. The top panel shows a schematic of the truncated Keap1 fragments. HEK293T cells that were cotransfected with myc-gankyrin and Flag-tagged truncated Keap1 fragments were lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibodies. (G) The interaction of Flag-KC (Kelch domain of Keap1) with Myc-tagged gankyrin. HEK293T cells cotransfected with Flag-KC and Myc-tagged gankyrin were immunoprecipitated with anti-flag antibody and immunoblotted with anti-myc antibodies. (H) The interaction of Flag-Keap1 with Myc-tagged gankyrin mutants. The top panel shows a schematic of the gankyrin mutants. HEK293T cells cotransfected with Flag-Keap1 and myc-tagged deletion mutants of gankyrin were immunoprecipitated with anti-flag antibody. Precipitated proteins and cell lysates were blotted with anti-myc and the indicated antibodies. (I) Wild-type or ExxE motif-mutated gankyrin and Flag-Keap1 plasmids were transfected into HEK293T cells, and the cells were then lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibody. N-mutated indicated E in aa 21-24 were mutated to A, C-mutated indicated E in aa 201-204 were mutated to A, and N+C-mutated indicated E in aa 21-24 and aa 201-204 were all mutated. (J) The knockdown of Keap1 abolished the regulatory role of gankyrin on Nrf2 protein levels. Negative control oligonucleotides or siRNA targeting Keap1 were transfected into MHCCLM3-Con, -siGank, or SMMC7721-Con, -ovGank cells. Cell lysates were blotted with anti-Nrf2 and other indicated antibodies. (K) A coimmunoprecipitation assay was used to analyze the amount of gankyrin that was associated with Keap1 after stimulation with sulforaphane, tBHQ, or H 2 O 2 . SMMC7721 cells were stimulated by sulforaphane, tBHQ, or H 2 O 2 for 12 h, and the cells were then lysed and immunoprecipitated with an anti-Keap1 antibody. Precipitates and cell lysates were blotted with an anti-gankyrin antibody. The data are representative of at least two experiments with similar results.
Figure Legend Snippet: Gankyrin binds to the Kelch domain of Keap1. (A) Gankyrin influenced the binding of Keap1 to Nrf2. Equal amounts of cell lysates were immunoprecipitated with an anti-Keap1 antibody. Precipitated proteins and cell lysates were blotted with anti-Nrf2, anti-gankyrin, and anti-Keap1 antibodies. (B) Confocal microscopy was performed on HEK293T cells cotransfected with Keap1 and myc-gankyrin. Bar, 10 µm. (C and D) Gankyrin and Keap1 were cotransfected into 293T cells. Whole cell lysates were immunoprecipitated with Keap1- (C) or gankyrin-specific (D) antibodies. Precipitated proteins and cell lysates were blotted with the indicated antibodies. (E) Cell lysates from SMMC7721-con and SMMC7721-ovGank cells were immunoprecipitated with anti-Keap1 antibodies, and Western blot analysis was performed with the indicated antibodies. (F) The interaction of Myc-gankyrin with Flag-tagged truncated Keap1 fragments. The top panel shows a schematic of the truncated Keap1 fragments. HEK293T cells that were cotransfected with myc-gankyrin and Flag-tagged truncated Keap1 fragments were lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibodies. (G) The interaction of Flag-KC (Kelch domain of Keap1) with Myc-tagged gankyrin. HEK293T cells cotransfected with Flag-KC and Myc-tagged gankyrin were immunoprecipitated with anti-flag antibody and immunoblotted with anti-myc antibodies. (H) The interaction of Flag-Keap1 with Myc-tagged gankyrin mutants. The top panel shows a schematic of the gankyrin mutants. HEK293T cells cotransfected with Flag-Keap1 and myc-tagged deletion mutants of gankyrin were immunoprecipitated with anti-flag antibody. Precipitated proteins and cell lysates were blotted with anti-myc and the indicated antibodies. (I) Wild-type or ExxE motif-mutated gankyrin and Flag-Keap1 plasmids were transfected into HEK293T cells, and the cells were then lysed and immunoprecipitated with anti-myc antibody. Precipitates and cell lysates were blotted with anti-Flag or anti-myc antibody. N-mutated indicated E in aa 21-24 were mutated to A, C-mutated indicated E in aa 201-204 were mutated to A, and N+C-mutated indicated E in aa 21-24 and aa 201-204 were all mutated. (J) The knockdown of Keap1 abolished the regulatory role of gankyrin on Nrf2 protein levels. Negative control oligonucleotides or siRNA targeting Keap1 were transfected into MHCCLM3-Con, -siGank, or SMMC7721-Con, -ovGank cells. Cell lysates were blotted with anti-Nrf2 and other indicated antibodies. (K) A coimmunoprecipitation assay was used to analyze the amount of gankyrin that was associated with Keap1 after stimulation with sulforaphane, tBHQ, or H 2 O 2 . SMMC7721 cells were stimulated by sulforaphane, tBHQ, or H 2 O 2 for 12 h, and the cells were then lysed and immunoprecipitated with an anti-Keap1 antibody. Precipitates and cell lysates were blotted with an anti-gankyrin antibody. The data are representative of at least two experiments with similar results.

Techniques Used: Binding Assay, Immunoprecipitation, Confocal Microscopy, Western Blot, Transfection, Negative Control, Co-Immunoprecipitation Assay

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

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-27123-8

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

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

Chicken SC protein expression. ( A ) Representative western blot analysis of protein bands in SCs exposed to 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S5 . Relative protein levels of ( B ) NRF2, KEAP1, PRDX4, ( C ) ATP5A, ( D ) p-AMPKα/AMPKα, and ( E ) p-mTOR/mTOR in SCs exposed to plasma. ( F ) Representative western blot analysis of protein bands in SCs trasfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S6 . Relative protein levels of ( G ) ATP5A, ( H ) p-AMPKα/AMPKα, and ( I ) p-mTOR/mTOR in transfected SCs. One independent replicate on western blot analysis of protein bands in SCs is presented in Supplementary Figure S7 . Data are represented as the mean ± SD (n = 3 per group). * p
Figure Legend Snippet: Chicken SC protein expression. ( A ) Representative western blot analysis of protein bands in SCs exposed to 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S5 . Relative protein levels of ( B ) NRF2, KEAP1, PRDX4, ( C ) ATP5A, ( D ) p-AMPKα/AMPKα, and ( E ) p-mTOR/mTOR in SCs exposed to plasma. ( F ) Representative western blot analysis of protein bands in SCs trasfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S6 . Relative protein levels of ( G ) ATP5A, ( H ) p-AMPKα/AMPKα, and ( I ) p-mTOR/mTOR in transfected SCs. One independent replicate on western blot analysis of protein bands in SCs is presented in Supplementary Figure S7 . Data are represented as the mean ± SD (n = 3 per group). * p

Techniques Used: Expressing, Western Blot, Transfection

22) Product Images from "Aromatase Inhibitor-Mediated Down Regulation of INrf2 (Keap1) Leads to Increased Nrf2 and Resistance in Breast Cancer"

Article Title: Aromatase Inhibitor-Mediated Down Regulation of INrf2 (Keap1) Leads to Increased Nrf2 and Resistance in Breast Cancer

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-14-0672

Tumor initiating cells (TIC) isolated from LTLTCa expressed lower levels of INrf2 and higher levels of Nrf2
Figure Legend Snippet: Tumor initiating cells (TIC) isolated from LTLTCa expressed lower levels of INrf2 and higher levels of Nrf2

Techniques Used: Isolation

Aromatase inhibitor-resistant cells generated increased levels of ROS and expressed lower levels of INrf2, higher levels of Nrf2 and Nrf2 downstream gene expression
Figure Legend Snippet: Aromatase inhibitor-resistant cells generated increased levels of ROS and expressed lower levels of INrf2, higher levels of Nrf2 and Nrf2 downstream gene expression

Techniques Used: Generated, Expressing

23) Product Images from "UHRF1 regulation of the Keap1–Nrf2 pathway in pancreatic cancer contributes to oncogenesis"

Article Title: UHRF1 regulation of the Keap1–Nrf2 pathway in pancreatic cancer contributes to oncogenesis

Journal: The Journal of Pathology

doi: 10.1002/path.4665

UHRF1 contributes to pancreatic cancer cell growth through activation of Nrf2. (A) GSH levels and (B, C) reactive oxygen species levels following the indicated treatments in MiaPaca‐2 cells ( n = 3). (D) Caspase 3/7 activity in Suit‐2 cells following the indicated treatments ( n = 3). (E, F) Western blot of cleaved caspase 3 and cleaved cyclin E following UHRF1 depletion from Suit‐2 cells ( n = 3 independent experiments). (G) MTS assay of MiaPaca‐2 cells 72 h after control treatments or depletion of UHRF1, Keap1 or both; U1 + K2 = UHRF1 siRNA1 + Keap1 siRNA2; U3 + K4 = UHRF1 siRNA3 + Keap1 siRNA4. (H) Western analysis of MiaPaca‐2 cells following the indicated treatments; *p
Figure Legend Snippet: UHRF1 contributes to pancreatic cancer cell growth through activation of Nrf2. (A) GSH levels and (B, C) reactive oxygen species levels following the indicated treatments in MiaPaca‐2 cells ( n = 3). (D) Caspase 3/7 activity in Suit‐2 cells following the indicated treatments ( n = 3). (E, F) Western blot of cleaved caspase 3 and cleaved cyclin E following UHRF1 depletion from Suit‐2 cells ( n = 3 independent experiments). (G) MTS assay of MiaPaca‐2 cells 72 h after control treatments or depletion of UHRF1, Keap1 or both; U1 + K2 = UHRF1 siRNA1 + Keap1 siRNA2; U3 + K4 = UHRF1 siRNA3 + Keap1 siRNA4. (H) Western analysis of MiaPaca‐2 cells following the indicated treatments; *p

Techniques Used: Activation Assay, Activity Assay, Western Blot, MTS Assay

UHRF1 suppression of Keap1 is required for efficient transition of pancreatic cancer cells through G 2 –M. (A) ICC of CFpac‐1 cells, showing variable UHRF1 expression. (B) UHRF1, cyclin E, A and B western blotting of CFpac‐1 cells/extracts sampled at the indicated time points, following release from a double thymidine block. (C) FACS profiles of PI‐stained MiaPaca‐2 cells following the indicated treatments. (D) Mean data plotted for two independent FACS experiments
Figure Legend Snippet: UHRF1 suppression of Keap1 is required for efficient transition of pancreatic cancer cells through G 2 –M. (A) ICC of CFpac‐1 cells, showing variable UHRF1 expression. (B) UHRF1, cyclin E, A and B western blotting of CFpac‐1 cells/extracts sampled at the indicated time points, following release from a double thymidine block. (C) FACS profiles of PI‐stained MiaPaca‐2 cells following the indicated treatments. (D) Mean data plotted for two independent FACS experiments

Techniques Used: Immunocytochemistry, Expressing, Western Blot, Blocking Assay, FACS, Staining

UHRF1 and Keap1 levels are inversely correlated in human PDAC tissues. (A) Immunohistochemistry of matched pancreatic cancer tissues stained for UHRF1 and Keap1. (B) The scores attributed for nuclear UHRF1 expression are plotted, grouped according to score and keap1 status; white bar, negative; black bar, positive. (C) Western blotting following K‐Ras knockdown
Figure Legend Snippet: UHRF1 and Keap1 levels are inversely correlated in human PDAC tissues. (A) Immunohistochemistry of matched pancreatic cancer tissues stained for UHRF1 and Keap1. (B) The scores attributed for nuclear UHRF1 expression are plotted, grouped according to score and keap1 status; white bar, negative; black bar, positive. (C) Western blotting following K‐Ras knockdown

Techniques Used: Immunohistochemistry, Staining, Expressing, Western Blot

UHRF1 regulates the Nrf2–Keap1 pathway by maintaining KEAP1 promoter methylation. (A) Basal methylation index detected by triplicate pyrosequencing of KEAP1 . (B) Mean of triplicate DNA methylation measurements of KEAP1 promoter in MiaPaca‐2, Suit2 and CFpac‐1 cells following control‐ or UHRF1‐targeting siRNAs. (C) RT–PCR for KEAP1 transcripts relative to GAPDH in control and UHRF1‐depleted Suit‐2 cells. (D, E) Western blot (Suit‐2) of the indicated proteins following UHRF1 or Nrf2 depletion. (F) RT–PCR for NRF2 transcripts relative to GAPDH in control and UHRF1‐ or Nrf2‐depleted Suit‐2 cells. (G, H) Gain in Keap1, down‐regulation of Nrf2 in MiaPaca‐2 and primary PDAC cells from a KPC mouse, respectively. (I) Relative luciferase activity from an 8× ARE‐reporter following UHRF1 or Nrf2 depletion (Suit‐2);** p
Figure Legend Snippet: UHRF1 regulates the Nrf2–Keap1 pathway by maintaining KEAP1 promoter methylation. (A) Basal methylation index detected by triplicate pyrosequencing of KEAP1 . (B) Mean of triplicate DNA methylation measurements of KEAP1 promoter in MiaPaca‐2, Suit2 and CFpac‐1 cells following control‐ or UHRF1‐targeting siRNAs. (C) RT–PCR for KEAP1 transcripts relative to GAPDH in control and UHRF1‐depleted Suit‐2 cells. (D, E) Western blot (Suit‐2) of the indicated proteins following UHRF1 or Nrf2 depletion. (F) RT–PCR for NRF2 transcripts relative to GAPDH in control and UHRF1‐ or Nrf2‐depleted Suit‐2 cells. (G, H) Gain in Keap1, down‐regulation of Nrf2 in MiaPaca‐2 and primary PDAC cells from a KPC mouse, respectively. (I) Relative luciferase activity from an 8× ARE‐reporter following UHRF1 or Nrf2 depletion (Suit‐2);** p

Techniques Used: Methylation, DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay

24) Product Images from "Dexmedetomidine Attenuates Orthotopic Liver Transplantation-Induced Acute Gut Injury via α2-Adrenergic Receptor-Dependent Suppression of Oxidative Stress"

Article Title: Dexmedetomidine Attenuates Orthotopic Liver Transplantation-Induced Acute Gut Injury via α2-Adrenergic Receptor-Dependent Suppression of Oxidative Stress

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2019/9426368

Silencing of α 2A -AR siRNA erased the protective role of Dex on antioxidation in IEC-6 cells with simulated H/R stimulation. (a) Summary of ROS levels in IEC-6 cells with different treatments. (b) Representative fluorescence immunostaining images of Nrf2 in IEC-6 cells with different treatments. Scale bar, 100 μ m. (c–d) Expression of Nrf2, NQO1, and Keap1 proteins and mRNA levels in intestinal tissues. (e–g) Summary of the relative expression levels of the transcripts for the genes HMOX1 (e), SOD1 (f), and CAT (g) in IEC-6 cells with different treatments, n = 6 for each group. A: control IEC-6 cells; B: IEC-6 cells with H/R treatment; C: IEC-6 cells that were pretreated with 1 nM Dex for 1 h before inducing H/R injury; D: IEC-6 cells with silencing of α 2A -AR siRNA; E: IEC-6 cells with silencing of α 2A -AR siRNA and H/R treatment; F: IEC-6 cells with silencing of α 2A -AR siRNA that were pretreated with Dex prior to H/R injury. ∗ P
Figure Legend Snippet: Silencing of α 2A -AR siRNA erased the protective role of Dex on antioxidation in IEC-6 cells with simulated H/R stimulation. (a) Summary of ROS levels in IEC-6 cells with different treatments. (b) Representative fluorescence immunostaining images of Nrf2 in IEC-6 cells with different treatments. Scale bar, 100 μ m. (c–d) Expression of Nrf2, NQO1, and Keap1 proteins and mRNA levels in intestinal tissues. (e–g) Summary of the relative expression levels of the transcripts for the genes HMOX1 (e), SOD1 (f), and CAT (g) in IEC-6 cells with different treatments, n = 6 for each group. A: control IEC-6 cells; B: IEC-6 cells with H/R treatment; C: IEC-6 cells that were pretreated with 1 nM Dex for 1 h before inducing H/R injury; D: IEC-6 cells with silencing of α 2A -AR siRNA; E: IEC-6 cells with silencing of α 2A -AR siRNA and H/R treatment; F: IEC-6 cells with silencing of α 2A -AR siRNA that were pretreated with Dex prior to H/R injury. ∗ P

Techniques Used: Fluorescence, Immunostaining, Expressing

25) 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

26) Product Images from "Dual TNFα-Induced Effects on NRF2 Mediated Antioxidant Defence in Astrocyte-Rich Cultures: Role of Protein Kinase Activation"

Article Title: Dual TNFα-Induced Effects on NRF2 Mediated Antioxidant Defence in Astrocyte-Rich Cultures: Role of Protein Kinase Activation

Journal: Neurochemical research

doi: 10.1007/s11064-012-0878-y

TNF α (24 h treatment) increased astroglial antioxidant defense system. Astrocyte-rich cultures were exposed to different concentrations of TNF α (0.5, 1 and 10 ng/mL) for 24 h followed by the protein expression analysis of Nrf2, Keap1,
Figure Legend Snippet: TNF α (24 h treatment) increased astroglial antioxidant defense system. Astrocyte-rich cultures were exposed to different concentrations of TNF α (0.5, 1 and 10 ng/mL) for 24 h followed by the protein expression analysis of Nrf2, Keap1,

Techniques Used: Expressing

TNF α (72 h treatment) decreased the astroglial antioxidant defense system. Astrocyte-rich cultures were exposed to different concentrations of TNF α (0.5, 1 and 10 ng/mL) for 72 h followed by protein expression analysis of Nrf2, Keap1,
Figure Legend Snippet: TNF α (72 h treatment) decreased the astroglial antioxidant defense system. Astrocyte-rich cultures were exposed to different concentrations of TNF α (0.5, 1 and 10 ng/mL) for 72 h followed by protein expression analysis of Nrf2, Keap1,

Techniques Used: Expressing

27) Product Images from "Chamomile Confers Protection against Hydrogen Peroxide-Induced Toxicity through Activation of Nrf2-Mediated Defense Response"

Article Title: Chamomile Confers Protection against Hydrogen Peroxide-Induced Toxicity through Activation of Nrf2-Mediated Defense Response

Journal: Phytotherapy research : PTR

doi: 10.1002/ptr.4701

Effect of chamomile on Nrf2/Keap1 expression in RAW 264.7 macrophages. (A) Western blot for cytosolic Nrf2 and Keap1 protein expression, and (B) nuclear expression of Nrf2 in RAW 264.7 macrophages stimulated with chamomile 0, 10, 20, and 40 μg/mL
Figure Legend Snippet: Effect of chamomile on Nrf2/Keap1 expression in RAW 264.7 macrophages. (A) Western blot for cytosolic Nrf2 and Keap1 protein expression, and (B) nuclear expression of Nrf2 in RAW 264.7 macrophages stimulated with chamomile 0, 10, 20, and 40 μg/mL

Techniques Used: Expressing, Western Blot

28) Product Images from "Anti-apoptotic effects of human placental hydrolysate against hepatocyte toxicity in vivo and in vitro"

Article Title: Anti-apoptotic effects of human placental hydrolysate against hepatocyte toxicity in vivo and in vitro

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3830

Upregulation of antioxidant enzymes and regulation of Keap1-Nrf2 in hPH-treated HepG2 cells. (A) Lysates from hPH-induced HepG2 cells were immunoblotted with anti-SOD-1, anti-SOD-2, anti-GPx, or anti-catalase antibodies. Representative images (left penal), densitometry results (right panel). All data are presented as the mean ± standard error of the mean. * P
Figure Legend Snippet: Upregulation of antioxidant enzymes and regulation of Keap1-Nrf2 in hPH-treated HepG2 cells. (A) Lysates from hPH-induced HepG2 cells were immunoblotted with anti-SOD-1, anti-SOD-2, anti-GPx, or anti-catalase antibodies. Representative images (left penal), densitometry results (right panel). All data are presented as the mean ± standard error of the mean. * P

Techniques Used:

Related Articles

Centrifugation:

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: Lysate was sonicated (Fisher Scientific FB-505), insoluble debris cleared by centrifugation, and the supernatant was diluted into 4X Laemmli buffer containing 50mM dithiothreitol (DTT) as a reducing agent. .. Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

Nucleic Acid Electrophoresis:

Article Title: Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels
Article Snippet: Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes via wet electrophoretic transfer (Bio-Rad, Hercules, California, USA). .. The following antibodies were used: anti-NRF2 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti-KEAP1 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti-PRDX4 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti- ATP5A (rabbit polyclonal; Abcam; 1:250), anti-phospho-AMPKα (Thr172, p-AMPKα; rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-AMPKα (rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-phospho-mTOR (Ser2448, p-mTOR; rabbit monoclonal; Cell Signaling Technology; 1:1,000), anti-mTOR (rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-beta actin (rabbit polyclonal; Bioss; 1:1,000).

Glutathione Assay:

Article Title: Cytotoxic Effect of Thymoquinone-Loaded Nanostructured Lipid Carrier (TQ-NLC) on Liver Cancer Cell Integrated with Hepatitis B Genome, Hep3B
Article Snippet: Other kits used were the Glutathione Assay Kit (Cayman, USA) and Annexin V/FITC Kit (BD Bioscience, USA). .. Primary rabbit antibodies anti-Nrf2 (C-20: sc-720), anti-Keap1 (H-190: sc-33569), caspase-3 (H-277: sc-7148), caspase-7 p20 (H-65: sc-33773), and anti-beta-actin (sc-47778) were purchased from Santa Cruz Biotechnology (CA, USA).

BIA-KA:

Article Title: Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress
Article Snippet: Western blotting Cell and tissue lysate were prepared as described previously [ ] and quantified by BCA assay. .. Protein was transferred to an Immobilon-P PVDF membrane (Millipore, Billerica, MA) and probed with anti-LC3-II (Novus Biologicals, Littleton, CO), anti-Beclin-1 (Novus Biologicals, Littleton, CO), anti-Atg7 (Sigma, St. Louis, MO), anti-p62 (BioLegend, San Diego, CA) or anti-Keap1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies.

Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis
Article Snippet: Proteins were quantified with the bicinchoninic acid assay (Micro BCA™ Protein Assay Kit; Thermo Scientific). .. The membranes were probed with the following primary antibodies: anti-GCLC (Thermo Scientific, #PA5–16581; 1:1000 dilution), anti-Keap1 (Santa Cruz, #33569; 1:1000 dilution), anti-p62 (R & D, #MAB8028; 1:250 dilution), anti-Nrf2 (Cell Signalling, #12721; 1:200 dilution), and anti-GAPDH (Santa Cruz, #sc25778; 1:5000 dilution, or sc-365062; 1:5000 dilution).

Pyrolysis Gas Chromatography:

Article Title: Resveratrol, an Nrf2 activator, ameliorates aging-related progressive renal injury
Article Snippet: .. Western blot analysis was performed using the following antibodies: Nrf2 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), Keap1 (Santa Cruz Biotechnology Inc.), Lamin B1 (Cell Signaling Technology Inc., Danvers, MA, USA), HO-1 (Cell Signaling Technology Inc.), NQO-1 (Santa Cruz Biotechnology Inc.), SIRT1 (Cell Signaling Technology Inc.), total AMPK (Cell Signaling Technology Inc.), phosphorylated (phospho)-Thr172 AMPK (Cell Signaling Technology Inc.), PPARα (Abcam), PGC-1α (Novus Biologicals, Littleton, CO, USA), estrogen-related receptor α (ERRα) (Millipore), SOD1 (Enzo Life Sciences, Farmingdale, NY, USA), SOD2 (Abcam), cytochrome c oxidase I (Santa Cruz Biotechnology) and IV (Cell Signaling Technology Inc.), B-cell leukaemia/lymphoma 2 (BCL-2) (Santa Cruz Biotechnology); BCL-2-associaated X protein (BAX) (Santa Cruz Biotechnology) and β-actin (1:10000, Sigma). .. Renal oxidative stress To evaluate oxidative stress, we measured the 24-h urinary 8-epi-prostaglandin F2α (isoprostane) and 24-h urinary 8-OH-dG concentrations using ELISA kits (OXIS Health Products Inc., Foster City, CA, USA).

Incubation:

Article Title: NRF2/miR-140 signaling confers radioprotection to human lung fibroblasts
Article Snippet: The membrane was incubated with specific primary antibody overnight followed by the horseradish peroxidase (HRP)-conjugated secondary antibody, and visualized by the ECL Western blotting detection system (Thermo Scientific; Rockford, IL). β-actin (Sigma; St Louis, MO, USA) was used as the loading control. .. Antibodies against BRCA1, NRF2 and KEAP1 were purchased from Santa Cruz (Santa Cruz Biotechnology; Dallas, TX).

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: For in situ compound or metabolite treatment experiments, compounds were added approximately 24 hours after transfection, and incubated for the indicated duration. .. Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

Article Title: Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels
Article Snippet: Membranes were blocked in PBS-0.08%Tween containing 5% dried skim milk or 3% BSA for 2 h at 25 °C, and subsequently incubated with primary antibodies at 4 °C overnight. .. The following antibodies were used: anti-NRF2 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti-KEAP1 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti-PRDX4 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti- ATP5A (rabbit polyclonal; Abcam; 1:250), anti-phospho-AMPKα (Thr172, p-AMPKα; rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-AMPKα (rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-phospho-mTOR (Ser2448, p-mTOR; rabbit monoclonal; Cell Signaling Technology; 1:1,000), anti-mTOR (rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-beta actin (rabbit polyclonal; Bioss; 1:1,000).

Article Title: Regulation of Nrf2 signaling and longevity in naturally long-lived rodents
Article Snippet: .. A total of 1,000 μg of protein in liver homogenates was incubated with Nrf2 (Santa Cruz) and Keap1 (Santa Cruz) after these antibodies were cross-linked to agarose beads. .. Protein interactions were analyzed via Immunoblot for βTrCP and Keap1 binding, respectively.

Expressing:

Article Title: NRF2/miR-140 signaling confers radioprotection to human lung fibroblasts
Article Snippet: Antibodies against BRCA1, NRF2 and KEAP1 were purchased from Santa Cruz (Santa Cruz Biotechnology; Dallas, TX). .. To determine the subcellular expression of proteins, cells were subjected to subcellular fractionation using the cell fractionation kit (Cell Signaling; Beverly, MA) according to the manufacturer’s protocol.

Article Title: Activation of Nrf2 by arsenite and monomethylarsonous acid is independent of Keap1-C151: enhanced Keap1-Cul3 interaction
Article Snippet: Antibodies against Nrf2 (Santa Cruz), Keap1 (Santa Cruz), chitin binding domain (New England Biolabs), α-tubulin (Santa Cruz), and the Myc and HA epitopes (Covance) were purchased from commercial sources. .. For detection of protein expression in total cell lysates, cells were lysed in sample buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% Glycerol, 100 mM dithiothreitol (DTT), 0.1% bromophenol blue).

Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis
Article Snippet: Paragraph title: Protein expression analysis ... The membranes were probed with the following primary antibodies: anti-GCLC (Thermo Scientific, #PA5–16581; 1:1000 dilution), anti-Keap1 (Santa Cruz, #33569; 1:1000 dilution), anti-p62 (R & D, #MAB8028; 1:250 dilution), anti-Nrf2 (Cell Signalling, #12721; 1:200 dilution), and anti-GAPDH (Santa Cruz, #sc25778; 1:5000 dilution, or sc-365062; 1:5000 dilution).

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: Paragraph title: FLAG-tagged Protein Expression and Western Blotting. ... Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

Article Title: Resveratrol, an Nrf2 activator, ameliorates aging-related progressive renal injury
Article Snippet: For Nrf2 expression, nuclear proteins were prepared using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Fisher Scientific, Rockford, IL, USA). .. Western blot analysis was performed using the following antibodies: Nrf2 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), Keap1 (Santa Cruz Biotechnology Inc.), Lamin B1 (Cell Signaling Technology Inc., Danvers, MA, USA), HO-1 (Cell Signaling Technology Inc.), NQO-1 (Santa Cruz Biotechnology Inc.), SIRT1 (Cell Signaling Technology Inc.), total AMPK (Cell Signaling Technology Inc.), phosphorylated (phospho)-Thr172 AMPK (Cell Signaling Technology Inc.), PPARα (Abcam), PGC-1α (Novus Biologicals, Littleton, CO, USA), estrogen-related receptor α (ERRα) (Millipore), SOD1 (Enzo Life Sciences, Farmingdale, NY, USA), SOD2 (Abcam), cytochrome c oxidase I (Santa Cruz Biotechnology) and IV (Cell Signaling Technology Inc.), B-cell leukaemia/lymphoma 2 (BCL-2) (Santa Cruz Biotechnology); BCL-2-associaated X protein (BAX) (Santa Cruz Biotechnology) and β-actin (1:10000, Sigma).

Cell Fractionation:

Article Title: NRF2/miR-140 signaling confers radioprotection to human lung fibroblasts
Article Snippet: Antibodies against BRCA1, NRF2 and KEAP1 were purchased from Santa Cruz (Santa Cruz Biotechnology; Dallas, TX). .. To determine the subcellular expression of proteins, cells were subjected to subcellular fractionation using the cell fractionation kit (Cell Signaling; Beverly, MA) according to the manufacturer’s protocol.

Modification:

Article Title: Cytotoxic Effect of Thymoquinone-Loaded Nanostructured Lipid Carrier (TQ-NLC) on Liver Cancer Cell Integrated with Hepatitis B Genome, Hep3B
Article Snippet: Dulbecco's Modified Eagle's Medium (DMEM), trypsin-EDTA, antibiotics (penicillin and streptomycin), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium bromide (MTT) powder, and trypan blue dye solution were purchased from Nacalai Tesque (Kyoto, Japan). .. Primary rabbit antibodies anti-Nrf2 (C-20: sc-720), anti-Keap1 (H-190: sc-33569), caspase-3 (H-277: sc-7148), caspase-7 p20 (H-65: sc-33773), and anti-beta-actin (sc-47778) were purchased from Santa Cruz Biotechnology (CA, USA).

Western Blot:

Article Title: Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression
Article Snippet: Paragraph title: Subcellular fractionation and western blotting ... Antibodies used in this study were as follows: anti-INrf2 (1:1000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cul3 (1:1000), anti-Rbx1 (1:1000) purchased from Cell Signaling (Danvers, MA), anti-V5 HRP (1:5000), anti-FLAG HRP purchased from Invitrogen, anti-phosphotyrosine (1:1000) and anti-actin (1:5000) purchased from Sigma-Aldrich (St Louis, MO).

Article Title: Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress
Article Snippet: Paragraph title: Western blotting ... Protein was transferred to an Immobilon-P PVDF membrane (Millipore, Billerica, MA) and probed with anti-LC3-II (Novus Biologicals, Littleton, CO), anti-Beclin-1 (Novus Biologicals, Littleton, CO), anti-Atg7 (Sigma, St. Louis, MO), anti-p62 (BioLegend, San Diego, CA) or anti-Keap1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies.

Article Title: Brd4 regulates the expression of essential autophagy genes and Keap1 in AML cells
Article Snippet: .. Antibodies used for Immunoblots: anti-LC3B ( #2775, Cell Signaling), anti-Keap1 (sc-365626, G-2, Santa Cruz Biotechnology), anti-Atg3 (sc-393660, A-3, Santa Cruz Biotechnology), anti-NQO1 (sc-393736, F-8, Santa Cruz Biotechnology), anti-GCLM (Ab124827, Abcam), anti-Bcl2 (sc-56015, 100/D5, Santa Cruz Biotechnology), anti- p62 (sc-25575, H-290, Santa Cruz Biotechnology), anti-actin (A5441, Sigma), and anti-Nrf2 (sc-13032, H-300, Santa Cruz Biotechnology), anti-Brd4 (A301-985A50, BETHYL Laboratories). .. Anti-CEBPβ (sc-7962, H-7, Santa Cruz Biotechnology).

Article Title: NRF2/miR-140 signaling confers radioprotection to human lung fibroblasts
Article Snippet: Paragraph title: Western blotting and subcellular fractionation ... Antibodies against BRCA1, NRF2 and KEAP1 were purchased from Santa Cruz (Santa Cruz Biotechnology; Dallas, TX).

Article Title: Delayed treatment with oleanolic acid attenuates tubulointerstitial fibrosis in chronic cyclosporine nephropathy through Nrf2/HO-1 signaling
Article Snippet: Paragraph title: Western blotting ... Specifically, proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and detected with the following antibody concentrations: Nrf2 (1:1000; Santa Cruz Biotechnology Inc, Texas, USA), Keap1 (1:1000; Santa Cruz Biotechnology Inc, Texas, USA), HO-1 (1:1000; BD Biosciences, California, USA), NQO1 (1:1000; Santa Cruz Biotechnology Inc, Texas, USA), Bcl-2 (1:500; Santa Cruz Biotechnology Inc, Texas, USA), Bax (1:500; Santa Cruz Biotechnology Inc, Texas, USA), SOD1 (1:5000; Assay Designs, MI, USA), SOD2 (1:10000; Abcam, Cambridge, UK), Catalase (1:2000; Abcam, Cambridge, UK), and β-actin (1:10000; Sigma-Aldrich, MO, USA).

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: Paragraph title: FLAG-tagged Protein Expression and Western Blotting. ... Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

Article Title: Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels
Article Snippet: Paragraph title: Western blotting ... The following antibodies were used: anti-NRF2 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti-KEAP1 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti-PRDX4 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti- ATP5A (rabbit polyclonal; Abcam; 1:250), anti-phospho-AMPKα (Thr172, p-AMPKα; rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-AMPKα (rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-phospho-mTOR (Ser2448, p-mTOR; rabbit monoclonal; Cell Signaling Technology; 1:1,000), anti-mTOR (rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-beta actin (rabbit polyclonal; Bioss; 1:1,000).

Article Title: Resveratrol, an Nrf2 activator, ameliorates aging-related progressive renal injury
Article Snippet: .. Western blot analysis was performed using the following antibodies: Nrf2 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), Keap1 (Santa Cruz Biotechnology Inc.), Lamin B1 (Cell Signaling Technology Inc., Danvers, MA, USA), HO-1 (Cell Signaling Technology Inc.), NQO-1 (Santa Cruz Biotechnology Inc.), SIRT1 (Cell Signaling Technology Inc.), total AMPK (Cell Signaling Technology Inc.), phosphorylated (phospho)-Thr172 AMPK (Cell Signaling Technology Inc.), PPARα (Abcam), PGC-1α (Novus Biologicals, Littleton, CO, USA), estrogen-related receptor α (ERRα) (Millipore), SOD1 (Enzo Life Sciences, Farmingdale, NY, USA), SOD2 (Abcam), cytochrome c oxidase I (Santa Cruz Biotechnology) and IV (Cell Signaling Technology Inc.), B-cell leukaemia/lymphoma 2 (BCL-2) (Santa Cruz Biotechnology); BCL-2-associaated X protein (BAX) (Santa Cruz Biotechnology) and β-actin (1:10000, Sigma). .. Renal oxidative stress To evaluate oxidative stress, we measured the 24-h urinary 8-epi-prostaglandin F2α (isoprostane) and 24-h urinary 8-OH-dG concentrations using ELISA kits (OXIS Health Products Inc., Foster City, CA, USA).

Transfection:

Article Title: Activation of Nrf2 by arsenite and monomethylarsonous acid is independent of Keap1-C151: enhanced Keap1-Cul3 interaction
Article Snippet: Antibodies against Nrf2 (Santa Cruz), Keap1 (Santa Cruz), chitin binding domain (New England Biolabs), α-tubulin (Santa Cruz), and the Myc and HA epitopes (Covance) were purchased from commercial sources. .. For experiments with transient transfection, cells were harvested 48 h post-transfection.

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: For in situ compound or metabolite treatment experiments, compounds were added approximately 24 hours after transfection, and incubated for the indicated duration. .. Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

Protease Inhibitor:

Article Title: Activation of Nrf2 by arsenite and monomethylarsonous acid is independent of Keap1-C151: enhanced Keap1-Cul3 interaction
Article Snippet: Antibodies against Nrf2 (Santa Cruz), Keap1 (Santa Cruz), chitin binding domain (New England Biolabs), α-tubulin (Santa Cruz), and the Myc and HA epitopes (Covance) were purchased from commercial sources. .. For immunoprecipitation assays, cells were lysed in RIPA buffer (10 mM sodium phosphate [pH 8.0], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail (Sigma).

Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis
Article Snippet: Protein expression analysis Proteins from frozen cirrhotic PBC liver tissues (n = 24) and controls (n = 16) were extracted with homogenization in an ice-cold RIPA buffer (50 mM Tris-HCl pH = 8, 150 mM NaCl, 1% NP-40, 0.5% NaDOC, 0.1% SDS, 1 mM EDTA, 100 mM PMSF, 100 mM NaF), which contained a protease inhibitor cocktail and PhosSTOP (Roche Diagnostics GmbH). .. The membranes were probed with the following primary antibodies: anti-GCLC (Thermo Scientific, #PA5–16581; 1:1000 dilution), anti-Keap1 (Santa Cruz, #33569; 1:1000 dilution), anti-p62 (R & D, #MAB8028; 1:250 dilution), anti-Nrf2 (Cell Signalling, #12721; 1:200 dilution), and anti-GAPDH (Santa Cruz, #sc25778; 1:5000 dilution, or sc-365062; 1:5000 dilution).

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: For FLAG-KEAP1 western blotting and immunoprecipitation experiments, cells were harvested by scraping, pelleted by centrifugation, washed twice with PBS and lysed in 8 M urea, 50 mM NH4 HCO3 , phosphatase inhibitor cocktail (Sigma Aldrich), and EDTA-free complete protease inhibitor (Roche), pH 8.0, at 4 ˚C. .. Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

Generated:

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling). .. Rabbit polyclonal anti-pgK antibody was generated using pgK-modified KLH and affinity purification as described at a 1:400 dilution of a 0.33 mg/mL stock in 10mM sodium HEPES (pH 7.5), 150mM NaCl, 30% glycerol and 0.02% sodium azide.

Imaging:

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling). .. Secondary donkey anti-rabbit, donkey anti-goat, and donkey anti-mouse (Licor), were used at 1:10,000 dilution in 2% BSA-containing TBST and incubated for 1 hour prior to washing and imaging on a Licor infrared scanner.

Protein Concentration:

Article Title: Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels
Article Snippet: Protein concentration was determined using a bicinchoninic acid protein assay kit (Sigma-Aldrich) using bovine serum albumin (BSA) as standard. .. The following antibodies were used: anti-NRF2 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti-KEAP1 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti-PRDX4 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti- ATP5A (rabbit polyclonal; Abcam; 1:250), anti-phospho-AMPKα (Thr172, p-AMPKα; rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-AMPKα (rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-phospho-mTOR (Ser2448, p-mTOR; rabbit monoclonal; Cell Signaling Technology; 1:1,000), anti-mTOR (rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-beta actin (rabbit polyclonal; Bioss; 1:1,000).

Sonication:

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: Lysate was sonicated (Fisher Scientific FB-505), insoluble debris cleared by centrifugation, and the supernatant was diluted into 4X Laemmli buffer containing 50mM dithiothreitol (DTT) as a reducing agent. .. Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

Affinity Purification:

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling). .. Rabbit polyclonal anti-pgK antibody was generated using pgK-modified KLH and affinity purification as described at a 1:400 dilution of a 0.33 mg/mL stock in 10mM sodium HEPES (pH 7.5), 150mM NaCl, 30% glycerol and 0.02% sodium azide.

Binding Assay:

Article Title: Activation of Nrf2 by arsenite and monomethylarsonous acid is independent of Keap1-C151: enhanced Keap1-Cul3 interaction
Article Snippet: .. Antibodies against Nrf2 (Santa Cruz), Keap1 (Santa Cruz), chitin binding domain (New England Biolabs), α-tubulin (Santa Cruz), and the Myc and HA epitopes (Covance) were purchased from commercial sources. .. For detection of protein expression in total cell lysates, cells were lysed in sample buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% Glycerol, 100 mM dithiothreitol (DTT), 0.1% bromophenol blue).

Article Title: Regulation of Nrf2 signaling and longevity in naturally long-lived rodents
Article Snippet: A total of 1,000 μg of protein in liver homogenates was incubated with Nrf2 (Santa Cruz) and Keap1 (Santa Cruz) after these antibodies were cross-linked to agarose beads. .. Protein interactions were analyzed via Immunoblot for βTrCP and Keap1 binding, respectively.

MTT Assay:

Article Title: Cytotoxic Effect of Thymoquinone-Loaded Nanostructured Lipid Carrier (TQ-NLC) on Liver Cancer Cell Integrated with Hepatitis B Genome, Hep3B
Article Snippet: Dulbecco's Modified Eagle's Medium (DMEM), trypsin-EDTA, antibiotics (penicillin and streptomycin), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium bromide (MTT) powder, and trypan blue dye solution were purchased from Nacalai Tesque (Kyoto, Japan). .. Primary rabbit antibodies anti-Nrf2 (C-20: sc-720), anti-Keap1 (H-190: sc-33569), caspase-3 (H-277: sc-7148), caspase-7 p20 (H-65: sc-33773), and anti-beta-actin (sc-47778) were purchased from Santa Cruz Biotechnology (CA, USA).

Isolation:

Article Title: Isodeoxyelephantopin induces protective autophagy in lung cancer cells via Nrf2-p62-keap1 feedback loop
Article Snippet: Reagents and chemicals ESI was isolated and purified in Professor Yao-Lan Li’s laboratory, College of Pharmacy, Jinan University, Guangzhou, China. .. Antibodies include anti-LC3A/B, anti-Beclin-1, anti-ATG3 from Cell Signaling Technology (Beverly, MA, USA), anti-p62 and anti-Keap1 from Santa Cruz (Santa Cruz, CA, USA), anti-Nrf2 and anti-ubiquitin from Abcam (Cambridge, UK).

Bicinchoninic Acid Protein Assay:

Article Title: Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels
Article Snippet: Protein concentration was determined using a bicinchoninic acid protein assay kit (Sigma-Aldrich) using bovine serum albumin (BSA) as standard. .. The following antibodies were used: anti-NRF2 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti-KEAP1 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti-PRDX4 (mouse monoclonal; Santa Cruz Biotechnology; 1:200), anti- ATP5A (rabbit polyclonal; Abcam; 1:250), anti-phospho-AMPKα (Thr172, p-AMPKα; rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-AMPKα (rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-phospho-mTOR (Ser2448, p-mTOR; rabbit monoclonal; Cell Signaling Technology; 1:1,000), anti-mTOR (rabbit polyclonal; Cell Signaling Technology; 1:1,000), anti-beta actin (rabbit polyclonal; Bioss; 1:1,000).

Purification:

Article Title: Isodeoxyelephantopin induces protective autophagy in lung cancer cells via Nrf2-p62-keap1 feedback loop
Article Snippet: Reagents and chemicals ESI was isolated and purified in Professor Yao-Lan Li’s laboratory, College of Pharmacy, Jinan University, Guangzhou, China. .. Antibodies include anti-LC3A/B, anti-Beclin-1, anti-ATG3 from Cell Signaling Technology (Beverly, MA, USA), anti-p62 and anti-Keap1 from Santa Cruz (Santa Cruz, CA, USA), anti-Nrf2 and anti-ubiquitin from Abcam (Cambridge, UK).

Protein Extraction:

Article Title: Delayed treatment with oleanolic acid attenuates tubulointerstitial fibrosis in chronic cyclosporine nephropathy through Nrf2/HO-1 signaling
Article Snippet: Western blotting For Western blot analysis, total protein of renal cortical tissues was extracted with a Pro-Prep Protein Extraction Solution (Intron Biotechnology, Gyeonggi-do, Korea) according to the manufacturer’s instructions. .. Specifically, proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and detected with the following antibody concentrations: Nrf2 (1:1000; Santa Cruz Biotechnology Inc, Texas, USA), Keap1 (1:1000; Santa Cruz Biotechnology Inc, Texas, USA), HO-1 (1:1000; BD Biosciences, California, USA), NQO1 (1:1000; Santa Cruz Biotechnology Inc, Texas, USA), Bcl-2 (1:500; Santa Cruz Biotechnology Inc, Texas, USA), Bax (1:500; Santa Cruz Biotechnology Inc, Texas, USA), SOD1 (1:5000; Assay Designs, MI, USA), SOD2 (1:10000; Abcam, Cambridge, UK), Catalase (1:2000; Abcam, Cambridge, UK), and β-actin (1:10000; Sigma-Aldrich, MO, USA).

Article Title: Resveratrol, an Nrf2 activator, ameliorates aging-related progressive renal injury
Article Snippet: Western blot analysis Total proteins from whole kidney tissues and HK2 cells were extracted using a Pro-Prep Protein Extraction Solution (Intron Biotechnology, Gyeonggi-Do, Republic of Korea) according to the manufacturer’s instructions. .. Western blot analysis was performed using the following antibodies: Nrf2 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), Keap1 (Santa Cruz Biotechnology Inc.), Lamin B1 (Cell Signaling Technology Inc., Danvers, MA, USA), HO-1 (Cell Signaling Technology Inc.), NQO-1 (Santa Cruz Biotechnology Inc.), SIRT1 (Cell Signaling Technology Inc.), total AMPK (Cell Signaling Technology Inc.), phosphorylated (phospho)-Thr172 AMPK (Cell Signaling Technology Inc.), PPARα (Abcam), PGC-1α (Novus Biologicals, Littleton, CO, USA), estrogen-related receptor α (ERRα) (Millipore), SOD1 (Enzo Life Sciences, Farmingdale, NY, USA), SOD2 (Abcam), cytochrome c oxidase I (Santa Cruz Biotechnology) and IV (Cell Signaling Technology Inc.), B-cell leukaemia/lymphoma 2 (BCL-2) (Santa Cruz Biotechnology); BCL-2-associaated X protein (BAX) (Santa Cruz Biotechnology) and β-actin (1:10000, Sigma).

SDS Page:

Article Title: NRF2/miR-140 signaling confers radioprotection to human lung fibroblasts
Article Snippet: Total cell lysates (15–50 µg) were separated by SDS–PAGE and blotted onto polyvinylidene difluoride membrane. .. Antibodies against BRCA1, NRF2 and KEAP1 were purchased from Santa Cruz (Santa Cruz Biotechnology; Dallas, TX).

Article Title: Delayed treatment with oleanolic acid attenuates tubulointerstitial fibrosis in chronic cyclosporine nephropathy through Nrf2/HO-1 signaling
Article Snippet: .. Specifically, proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and detected with the following antibody concentrations: Nrf2 (1:1000; Santa Cruz Biotechnology Inc, Texas, USA), Keap1 (1:1000; Santa Cruz Biotechnology Inc, Texas, USA), HO-1 (1:1000; BD Biosciences, California, USA), NQO1 (1:1000; Santa Cruz Biotechnology Inc, Texas, USA), Bcl-2 (1:500; Santa Cruz Biotechnology Inc, Texas, USA), Bax (1:500; Santa Cruz Biotechnology Inc, Texas, USA), SOD1 (1:5000; Assay Designs, MI, USA), SOD2 (1:10000; Abcam, Cambridge, UK), Catalase (1:2000; Abcam, Cambridge, UK), and β-actin (1:10000; Sigma-Aldrich, MO, USA). ..

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: Samples were prepared for SDS-PAGE by heating to 95 ˚C for 5 minutes, cooled to room temperature, resolved on NuPAGE Novex 4-12% Bis-Tris Protein Gels (Invitrogen), and transferred onto nitrocellulose membranes by standard western blotting methods. .. Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

Plasmid Preparation:

Article Title: Time-dependent effects of systemic lipopolysaccharide injection on regulators of antioxidant defense Nrf2 and PGC-1 ? in neonatal rat brain.
Article Snippet: Anti-Keap1, anti-PGC-1α, anti-actin, anti-α-tubulin, anti-γGCL-C and anti-γGCL-M antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany). .. Anti-Keap1, anti-PGC-1α, anti-actin, anti-α-tubulin, anti-γGCL-C and anti-γGCL-M antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany).

Software:

Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis
Article Snippet: The membranes were probed with the following primary antibodies: anti-GCLC (Thermo Scientific, #PA5–16581; 1:1000 dilution), anti-Keap1 (Santa Cruz, #33569; 1:1000 dilution), anti-p62 (R & D, #MAB8028; 1:250 dilution), anti-Nrf2 (Cell Signalling, #12721; 1:200 dilution), and anti-GAPDH (Santa Cruz, #sc25778; 1:5000 dilution, or sc-365062; 1:5000 dilution). .. Bands were visualized and quantified with the MicroChemi 2.0 System and GelQuant software (Israel).

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling). .. Densitometry measurements were performed with ImageJ software.

In Situ:

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: For in situ compound or metabolite treatment experiments, compounds were added approximately 24 hours after transfection, and incubated for the indicated duration. .. Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

Homogenization:

Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis
Article Snippet: Protein expression analysis Proteins from frozen cirrhotic PBC liver tissues (n = 24) and controls (n = 16) were extracted with homogenization in an ice-cold RIPA buffer (50 mM Tris-HCl pH = 8, 150 mM NaCl, 1% NP-40, 0.5% NaDOC, 0.1% SDS, 1 mM EDTA, 100 mM PMSF, 100 mM NaF), which contained a protease inhibitor cocktail and PhosSTOP (Roche Diagnostics GmbH). .. The membranes were probed with the following primary antibodies: anti-GCLC (Thermo Scientific, #PA5–16581; 1:1000 dilution), anti-Keap1 (Santa Cruz, #33569; 1:1000 dilution), anti-p62 (R & D, #MAB8028; 1:250 dilution), anti-Nrf2 (Cell Signalling, #12721; 1:200 dilution), and anti-GAPDH (Santa Cruz, #sc25778; 1:5000 dilution, or sc-365062; 1:5000 dilution).

Acid Assay:

Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis
Article Snippet: Proteins were quantified with the bicinchoninic acid assay (Micro BCA™ Protein Assay Kit; Thermo Scientific). .. The membranes were probed with the following primary antibodies: anti-GCLC (Thermo Scientific, #PA5–16581; 1:1000 dilution), anti-Keap1 (Santa Cruz, #33569; 1:1000 dilution), anti-p62 (R & D, #MAB8028; 1:250 dilution), anti-Nrf2 (Cell Signalling, #12721; 1:200 dilution), and anti-GAPDH (Santa Cruz, #sc25778; 1:5000 dilution, or sc-365062; 1:5000 dilution).

Immunoprecipitation:

Article Title: Activation of Nrf2 by arsenite and monomethylarsonous acid is independent of Keap1-C151: enhanced Keap1-Cul3 interaction
Article Snippet: Paragraph title: Antibodies, immunoprecipitation, and immunoblot analysis ... Antibodies against Nrf2 (Santa Cruz), Keap1 (Santa Cruz), chitin binding domain (New England Biolabs), α-tubulin (Santa Cruz), and the Myc and HA epitopes (Covance) were purchased from commercial sources.

Article Title: A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling
Article Snippet: For FLAG-KEAP1 western blotting and immunoprecipitation experiments, cells were harvested by scraping, pelleted by centrifugation, washed twice with PBS and lysed in 8 M urea, 50 mM NH4 HCO3 , phosphatase inhibitor cocktail (Sigma Aldrich), and EDTA-free complete protease inhibitor (Roche), pH 8.0, at 4 ˚C. .. Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

Article Title: Regulation of Nrf2 signaling and longevity in naturally long-lived rodents
Article Snippet: Paragraph title: Immunoprecipitation. ... A total of 1,000 μg of protein in liver homogenates was incubated with Nrf2 (Santa Cruz) and Keap1 (Santa Cruz) after these antibodies were cross-linked to agarose beads.

Fractionation:

Article Title: Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression
Article Snippet: Paragraph title: Subcellular fractionation and western blotting ... Antibodies used in this study were as follows: anti-INrf2 (1:1000) purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cul3 (1:1000), anti-Rbx1 (1:1000) purchased from Cell Signaling (Danvers, MA), anti-V5 HRP (1:5000), anti-FLAG HRP purchased from Invitrogen, anti-phosphotyrosine (1:1000) and anti-actin (1:5000) purchased from Sigma-Aldrich (St Louis, MO).

Article Title: NRF2/miR-140 signaling confers radioprotection to human lung fibroblasts
Article Snippet: Paragraph title: Western blotting and subcellular fractionation ... Antibodies against BRCA1, NRF2 and KEAP1 were purchased from Santa Cruz (Santa Cruz Biotechnology; Dallas, TX).

Lysis:

Article Title: Brd4 regulates the expression of essential autophagy genes and Keap1 in AML cells
Article Snippet: Western blot Cell lysis and western blot were performed as described previously (Huang M, Leukemia 2013). .. Antibodies used for Immunoblots: anti-LC3B ( #2775, Cell Signaling), anti-Keap1 (sc-365626, G-2, Santa Cruz Biotechnology), anti-Atg3 (sc-393660, A-3, Santa Cruz Biotechnology), anti-NQO1 (sc-393736, F-8, Santa Cruz Biotechnology), anti-GCLM (Ab124827, Abcam), anti-Bcl2 (sc-56015, 100/D5, Santa Cruz Biotechnology), anti- p62 (sc-25575, H-290, Santa Cruz Biotechnology), anti-actin (A5441, Sigma), and anti-Nrf2 (sc-13032, H-300, Santa Cruz Biotechnology), anti-Brd4 (A301-985A50, BETHYL Laboratories).

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  • 99
    Santa Cruz Biotechnology anti keap1
    Schematic of SILAC-based proteomic mapping of <t>KEAP1</t> modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.
    Anti Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 8 article reviews
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    anti keap1 - by Bioz Stars, 2020-02
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    99
    Santa Cruz Biotechnology rabbit anti keap1
    Liver expression of Nrf2, <t>Keap1</t> and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.
    Rabbit Anti Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti keap1/product/Santa Cruz Biotechnology
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    rabbit anti keap1 - by Bioz Stars, 2020-02
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    Schematic of SILAC-based proteomic mapping of KEAP1 modifications in response to CBR-470-1 and NMR characterization of CR-MGx peptide. a, Stable isotope-labeled cells (stable isotope labeling with amino acids in cell culture, SILAC) expressing FLAG-tagged KEAP1 were treated with vehicle (‘light’) and CBR-470-1 or MGx (‘heavy’), respectively. Subsequent mixing of the cell lysates, anti-FLAG enrichment, tryptic digestion and LC-MS/MS analysis permitted detection of unmodified portions of KEAP1, which retained ∼1:1 SILAC ratios relative to the median ratios for all detected KEAP1 peptides. In contrast, peptides that are modified under one condition will no longer match tryptic MS/MS searches, resulting skewed SILAC ratios that “drop out” (bottom). b, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched DMSO treated ‘light’ cells and CBR-470-1 treated ‘heavy’ cells, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 3- to 4-fold upon relative to the KEAP1 median, indicative of structural modification ( n =8). c, Structural depiction of potentially modified stretches of human KEAP1 (red) using published x-ray crystal structure of the BTB (PDB: 4CXI) and KELCH (PDB: 1U6D) domains. Intervening protein stretches are depicted as unstructured loops in green. d, SILAC ratios for individual tryptic peptides from FLAG-KEAP1 enriched MGx treated ‘heavy’ cell lysates and no treated ‘light’ cell lysates, relative to the median ratio of all KEAP1 peptides. Highlighted tryptic peptides were significantly reduced by 2- to 2.5- fold upon relative to the KEAP1 median, indicative of structural modification ( n =12). e, Representative Western blotting analysis of FLAG-KEAP1 dimerization from HEK293T cells pre-treated with Bardoxolone methyl followed by CBR-470-1 treatment for 4 hours ( n =3). f, 1 H-NMR of CR-MGx peptide (isolated product of MGx incubated with Ac-NH-VVCGGGRGG-C(O)NH 2 peptide). 1 H NMR (500MHz, d6-DMSO) δ 12.17 (s, 1H), 12.02 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.23 (t, J = 5.6 Hz, 1H), 8.14 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 4.33-4.28 (m, 1H), 4.25-4.16 (m, 3H), 3.83 (dd, J = 6.9 Hz, J = 16.2 Hz, 1H), 3.79-3.67 (m, 6H), 3.63 (d, J = 5.7 Hz, 2H), 3.54 (dd, J = 4.9 Hz, J = 16.2 Hz, 1H), 3.18-3.13 (m, 2H), 3.04 (dd, J = 4.9 Hz, J = 13.9 Hz, 1H), 2.88 (dd, J = 8.6 Hz, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.96 (sep, J = 6.8 Hz, 2H), 1.87 (s, 3H), 1.80-1.75 (m, 1H), 1.56-1.47 (m, 3H), .87-.82 (m, 12H). g, 1 H-NMR of CR peptide (Ac-NH-VVCGGGRGG-C(O)NH 2 ). 1 H NMR (500MHz, d6-DMSO) δ 8.27-8.24 (m, 2H), 8.18 (t, J = 5.7 Hz, 1H), 8.13-8.08 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.91 (d, J = 8.8 Hz), 7.86 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 4.39 (dt, J = 5.6 Hz, J = 7.4 Hz, 1H), 4.28 (dt, J = 5.7 Hz, J = 7.2 Hz, 1H), 4.21-4.13 (m, 2H), 3.82-3.70 (m, 8H), 3.64 (d, J = 5.8, 2H), 3.08 (dt, J = 6.5 Hz, J = 6.5 Hz, 2H), 2.80-2.67 (m, 2H), 2.43 (t, J = 8.6 Hz, 1H), 1.94 (sep, J = 6.8 Hz, 2H), 1.85 (s, 3H), 1.75-1.68 (m, 1H), 1.54-1.42 (m, 3H), .85-.81 (m, 12H) h, 1 H- 1 H TOCSY of CR-MGx peptide. i, Peak assignment for CR-MGx peptide TOCSY spectrum. Data are mean ± SEM of biologically independent samples.

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

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

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

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

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

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

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

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

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

    Journal: Nature

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

    doi: 10.1038/s41586-018-0622-0

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

    Article Snippet: Primary antibodies used in this study include: anti-FLAG-M2 (1:1000, F1804, Sigma Aldrich), anti-KEAP1 (1:500, SC-15246, Santa Cruz), anti-HSPA1A (1:1000, 4872, Cell Signaling), anti-ACTB (1:1000, 4790, Cell Signaling), anti-GAPDH (1:1000, 2118S, Cell Signaling) and TUBG (1:1000, 5886, Cell Signaling).

    Techniques:

    Depletion of Keap1 reduces MitoQ-induced autophagy and increases transcriptional activity of the antioxidant Nrf2 (A) MDA-MB-231 cells were treated with increasing concentrations of siRNA oligonucleotides for 24 hr to optimize the downregulation of Keap1. Cells were treated with 10 nM Keap1 siRNA or control NTP siRNA for 24 hr before being treated with MitoQ (1 or 5 μM) for 24 hr. (B) LC3-II protein was used as an autophagy marker. Rapamycin (10 μM) was used as a positive control. (C) Autophagic flux was determined by treating cells with the lysosomal protease inhibitors Pepstatin A (10 μg/ml) and E64d (10 μg/ml) in the presence or absence of MitoQ (1 or 5 μM) for 24 hr. (D) The transcriptional activity of Nrf2 was measured with an assay with immobilized oligonucleotide containing the ARE consensus binding site. tBHQ (20 μM) was used as a positive control. Error bars represent S.D. *statistical significance compared with NTP siRNA cells. (E) Autophagy impairment was measured by observing levels of the autophagy substrate p62.

    Journal: Oncotarget

    Article Title: Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress

    doi:

    Figure Lengend Snippet: Depletion of Keap1 reduces MitoQ-induced autophagy and increases transcriptional activity of the antioxidant Nrf2 (A) MDA-MB-231 cells were treated with increasing concentrations of siRNA oligonucleotides for 24 hr to optimize the downregulation of Keap1. Cells were treated with 10 nM Keap1 siRNA or control NTP siRNA for 24 hr before being treated with MitoQ (1 or 5 μM) for 24 hr. (B) LC3-II protein was used as an autophagy marker. Rapamycin (10 μM) was used as a positive control. (C) Autophagic flux was determined by treating cells with the lysosomal protease inhibitors Pepstatin A (10 μg/ml) and E64d (10 μg/ml) in the presence or absence of MitoQ (1 or 5 μM) for 24 hr. (D) The transcriptional activity of Nrf2 was measured with an assay with immobilized oligonucleotide containing the ARE consensus binding site. tBHQ (20 μM) was used as a positive control. Error bars represent S.D. *statistical significance compared with NTP siRNA cells. (E) Autophagy impairment was measured by observing levels of the autophagy substrate p62.

    Article Snippet: Protein was transferred to an Immobilon-P PVDF membrane (Millipore, Billerica, MA) and probed with anti-LC3-II (Novus Biologicals, Littleton, CO), anti-Beclin-1 (Novus Biologicals, Littleton, CO), anti-Atg7 (Sigma, St. Louis, MO), anti-p62 (BioLegend, San Diego, CA) or anti-Keap1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies.

    Techniques: Activity Assay, Multiple Displacement Amplification, Marker, Positive Control, Binding Assay

    Depletion of Atg7 inhibits Keap1 degradation in breast cancer cells and MEF (A) MDA-MB-231 cells were transfected with 10 nM Atg7 siRNA or control NTP siRNA for 48 hr before MitoQ treatment. MDA-MB-231 cells were treated with 5 μM of MitoQ, and (B) Atg7 +/+ or Atg7 −/− MEF cells were treated with 5 μM MitoQ. Following 2, 6, or 24 hr of drug exposure, Keap1 degradation was analyzed by Western blot. 50μM tBHQ was used as a positive control. Error bars represent S.D. *statistical significance (p

    Journal: Oncotarget

    Article Title: Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress

    doi:

    Figure Lengend Snippet: Depletion of Atg7 inhibits Keap1 degradation in breast cancer cells and MEF (A) MDA-MB-231 cells were transfected with 10 nM Atg7 siRNA or control NTP siRNA for 48 hr before MitoQ treatment. MDA-MB-231 cells were treated with 5 μM of MitoQ, and (B) Atg7 +/+ or Atg7 −/− MEF cells were treated with 5 μM MitoQ. Following 2, 6, or 24 hr of drug exposure, Keap1 degradation was analyzed by Western blot. 50μM tBHQ was used as a positive control. Error bars represent S.D. *statistical significance (p

    Article Snippet: Protein was transferred to an Immobilon-P PVDF membrane (Millipore, Billerica, MA) and probed with anti-LC3-II (Novus Biologicals, Littleton, CO), anti-Beclin-1 (Novus Biologicals, Littleton, CO), anti-Atg7 (Sigma, St. Louis, MO), anti-p62 (BioLegend, San Diego, CA) or anti-Keap1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies.

    Techniques: Multiple Displacement Amplification, Transfection, Western Blot, Positive Control

    Effects of XXT on Nrf2 and Keap1 mRNA expression levels in HUVECs.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    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

    doi: 10.1155/2015/187265

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

    Article Snippet: Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing

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

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    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

    doi: 10.1155/2015/187265

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

    Article Snippet: Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing

    Schematic representation of XXT activities on Keap1-Nrf2-ARE pathway.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    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

    doi: 10.1155/2015/187265

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

    Article Snippet: Anti-Nrf2, Keap1, GCLM, NQO1, HMOX1, and anti-Keap1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques:

    Liver expression of Nrf2, Keap1 and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.

    Journal: Scientific Reports

    Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis

    doi: 10.1038/srep44769

    Figure Lengend Snippet: Liver expression of Nrf2, Keap1 and CK19 proteins in patients with cirrhotic PBC and controls. Representative immunohistochemical staining of Nrf2 ( A,B,C,J,K,L ), Keap1 ( D,E,F,M,N,O ) and CK19 ( G,H,I,P,Q,R ) proteins in serial sections of liver tissue from healthy controls (A–I) and cirrhotic PBC (J–R) . In healthy tissue, CK19-positive cells are marked by arrow (large bile duct) or arrowhead (small bile duct). In sections of cirrhotic livers, the corresponding areas are labelled by asterisks. Nrf2 was present only in fibrotic areas (J,K,L), in contrast to Keap1 which was expressed in fibrotic areas as well as in nodules (M,N,O). Original magnification 200x or 400x.

    Article Snippet: Then sections were probed with rabbit anti-Keap1 (Santa Cruz, #33569; 1:50 dilution), anti-Nrf2 (Cell Signaling, # 12721 S; 1:20 dilution), anti-CK19 (Santa Cruz, #33119; 1:50 dilution).

    Techniques: Expressing, Immunohistochemistry, Staining

    The hepatic expression of Keap1 in liver tissues of patients with PBC and controls. ( A ) Keap1 protein levels were determined with densitometry analyses, after normalization to GAPDH as a loading control. ( B ) Keap1 mRNA levels were estimated in patients with cirrhotic PBC, patients with early stage PBC, and controls. Results were normalized to 18sRNA. Bars indicate the mean ± SEM. ( C ) Representative immunofluorescence micrographs show liver sections from patients with PBC. (a) Nuclei are stained with DAPI (blue). (b) Immunofluorescence staining of Keap1 (green) shows its abundance in hepatocytes. (c) Arrows indicate the perinuclear and nuclear localizations of Keap1whereas arrowheads indicate cytoplasmic localization of Keap1.

    Journal: Scientific Reports

    Article Title: Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis

    doi: 10.1038/srep44769

    Figure Lengend Snippet: The hepatic expression of Keap1 in liver tissues of patients with PBC and controls. ( A ) Keap1 protein levels were determined with densitometry analyses, after normalization to GAPDH as a loading control. ( B ) Keap1 mRNA levels were estimated in patients with cirrhotic PBC, patients with early stage PBC, and controls. Results were normalized to 18sRNA. Bars indicate the mean ± SEM. ( C ) Representative immunofluorescence micrographs show liver sections from patients with PBC. (a) Nuclei are stained with DAPI (blue). (b) Immunofluorescence staining of Keap1 (green) shows its abundance in hepatocytes. (c) Arrows indicate the perinuclear and nuclear localizations of Keap1whereas arrowheads indicate cytoplasmic localization of Keap1.

    Article Snippet: Then sections were probed with rabbit anti-Keap1 (Santa Cruz, #33569; 1:50 dilution), anti-Nrf2 (Cell Signaling, # 12721 S; 1:20 dilution), anti-CK19 (Santa Cruz, #33119; 1:50 dilution).

    Techniques: Expressing, Immunofluorescence, Staining