Structured Review

Santa Cruz Biotechnology anti inrf2
Cul3–Rbx1 export out of the nucleus by means of the <t>INrf2</t> nuclear export signal. ( A , B ) Immunocytochemistry: HepG2 cells (A) or Hepa-1 cells (B) were grown in Lab-Tek II chamber slides. Cells were fixed, permeabilized and incubated with a 1:500 dilution of anti-goat INrf2, anti-rabbit Cul3 and anti-rabbit Rbx1 antibody, as indicated in the figures. Cells were washed and incubated with Alexa-Fluor-594-conjugated anti-goat antibody or FITC-conjugated anti-rabbit secondary antibody (Invitrogen). Cells were washed twice with PBS, stained with Vectashield containing nuclear DAPI stain, observed under a Nikon fluorescence microscope and photographed. ( C ) Hepa-1 cells were transfected with INrf2 siRNA for 48 hours. Cells were then treated with 100 μM t -BHQ for the indicated periods of time and harvested. Cytosolic and nuclear extracts were prepared, and lysates were immunoblotted with antibodies against INrf2, Cul3, Rbx1, LDH and lamin B. ( D ) Schematic diagram of the mouse INrf2 gene showing the BTB domain (responsible for Cul3 interaction), DGR domain (responsible for Nrf2 interaction) and a functional nuclear export signal (NES). ( E ) HepG2 cells were treated and transfected with 1 μg of vector encoding INrf2ΔNES–V5. Cells were then treated with 100 μM t -BHQ, harvested and nuclear and cytosolic extracts were then prepared. Lysates were immunoblotted with antibodies against V5, Cul3, Rbx1, lamin B and LDH. ( F ) HepG2 cells were co-transfected with 1 μg of INrf2–V5 or INrf2Y85A–V5 and 1 μg of FLAG–Crm1 and then treated with either DMSO or 100 μM t -BHQ for the indicated periods of time (left and right panels). Cells were harvested; 1 mg of lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to FLAG and V5 (top two panels). 1 mg of lysate was immunoprecipitated with antibody to FLAG and western blotted with antibodies to V5 and FLAG (bottom two panels).
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1) Product Images from "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 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

Journal: Journal of Cell Science

doi: 10.1242/jcs.097295

Cul3–Rbx1 export out of the nucleus by means of the INrf2 nuclear export signal. ( A , B ) Immunocytochemistry: HepG2 cells (A) or Hepa-1 cells (B) were grown in Lab-Tek II chamber slides. Cells were fixed, permeabilized and incubated with a 1:500 dilution of anti-goat INrf2, anti-rabbit Cul3 and anti-rabbit Rbx1 antibody, as indicated in the figures. Cells were washed and incubated with Alexa-Fluor-594-conjugated anti-goat antibody or FITC-conjugated anti-rabbit secondary antibody (Invitrogen). Cells were washed twice with PBS, stained with Vectashield containing nuclear DAPI stain, observed under a Nikon fluorescence microscope and photographed. ( C ) Hepa-1 cells were transfected with INrf2 siRNA for 48 hours. Cells were then treated with 100 μM t -BHQ for the indicated periods of time and harvested. Cytosolic and nuclear extracts were prepared, and lysates were immunoblotted with antibodies against INrf2, Cul3, Rbx1, LDH and lamin B. ( D ) Schematic diagram of the mouse INrf2 gene showing the BTB domain (responsible for Cul3 interaction), DGR domain (responsible for Nrf2 interaction) and a functional nuclear export signal (NES). ( E ) HepG2 cells were treated and transfected with 1 μg of vector encoding INrf2ΔNES–V5. Cells were then treated with 100 μM t -BHQ, harvested and nuclear and cytosolic extracts were then prepared. Lysates were immunoblotted with antibodies against V5, Cul3, Rbx1, lamin B and LDH. ( F ) HepG2 cells were co-transfected with 1 μg of INrf2–V5 or INrf2Y85A–V5 and 1 μg of FLAG–Crm1 and then treated with either DMSO or 100 μM t -BHQ for the indicated periods of time (left and right panels). Cells were harvested; 1 mg of lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to FLAG and V5 (top two panels). 1 mg of lysate was immunoprecipitated with antibody to FLAG and western blotted with antibodies to V5 and FLAG (bottom two panels).
Figure Legend Snippet: Cul3–Rbx1 export out of the nucleus by means of the INrf2 nuclear export signal. ( A , B ) Immunocytochemistry: HepG2 cells (A) or Hepa-1 cells (B) were grown in Lab-Tek II chamber slides. Cells were fixed, permeabilized and incubated with a 1:500 dilution of anti-goat INrf2, anti-rabbit Cul3 and anti-rabbit Rbx1 antibody, as indicated in the figures. Cells were washed and incubated with Alexa-Fluor-594-conjugated anti-goat antibody or FITC-conjugated anti-rabbit secondary antibody (Invitrogen). Cells were washed twice with PBS, stained with Vectashield containing nuclear DAPI stain, observed under a Nikon fluorescence microscope and photographed. ( C ) Hepa-1 cells were transfected with INrf2 siRNA for 48 hours. Cells were then treated with 100 μM t -BHQ for the indicated periods of time and harvested. Cytosolic and nuclear extracts were prepared, and lysates were immunoblotted with antibodies against INrf2, Cul3, Rbx1, LDH and lamin B. ( D ) Schematic diagram of the mouse INrf2 gene showing the BTB domain (responsible for Cul3 interaction), DGR domain (responsible for Nrf2 interaction) and a functional nuclear export signal (NES). ( E ) HepG2 cells were treated and transfected with 1 μg of vector encoding INrf2ΔNES–V5. Cells were then treated with 100 μM t -BHQ, harvested and nuclear and cytosolic extracts were then prepared. Lysates were immunoblotted with antibodies against V5, Cul3, Rbx1, lamin B and LDH. ( F ) HepG2 cells were co-transfected with 1 μg of INrf2–V5 or INrf2Y85A–V5 and 1 μg of FLAG–Crm1 and then treated with either DMSO or 100 μM t -BHQ for the indicated periods of time (left and right panels). Cells were harvested; 1 mg of lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to FLAG and V5 (top two panels). 1 mg of lysate was immunoprecipitated with antibody to FLAG and western blotted with antibodies to V5 and FLAG (bottom two panels).

Techniques Used: Immunocytochemistry, Incubation, Staining, Fluorescence, Microscopy, Transfection, Functional Assay, Plasmid Preparation, Immunoprecipitation, Western Blot

Immunoprecipitation of Cul3 and Rbx1. ( A , B ) HepG2 cells were treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were then collected, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody to (A) Cul3 and (B) Rbx1 and western blotted with antibodies to phosphotyrosine and INrf2 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibody to Cul3, Rbx1 and phosphotyrosine (bottom panels). ( C , D ) HepG2 cells were transfected with 1 μg of Cul3–V5 (C, left panel), Rbx1–V5 (D, left panel), Cul3Y764A–V5 (C, right panel) or Rbx1Y106–V5 (D, right panel) and treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were harvested, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to phosphotyrosine and V5 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibody to V5 (bottom panels). Asterisks (*) indicate the Rbx1 bands.
Figure Legend Snippet: Immunoprecipitation of Cul3 and Rbx1. ( A , B ) HepG2 cells were treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were then collected, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody to (A) Cul3 and (B) Rbx1 and western blotted with antibodies to phosphotyrosine and INrf2 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibody to Cul3, Rbx1 and phosphotyrosine (bottom panels). ( C , D ) HepG2 cells were transfected with 1 μg of Cul3–V5 (C, left panel), Rbx1–V5 (D, left panel), Cul3Y764A–V5 (C, right panel) or Rbx1Y106–V5 (D, right panel) and treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were harvested, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to phosphotyrosine and V5 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibody to V5 (bottom panels). Asterisks (*) indicate the Rbx1 bands.

Techniques Used: Immunoprecipitation, Western Blot, Transfection

Subcellular localization of mutant forms of INrf2. ( A – D ) HepG2 cells were transfected with 1 μg of mutant plasmids encoding INrf2Y85A, INrf2Y141A, INrf2Y208A or INrf2Y255A. Cells were then treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were harvested and nuclear and cytosolic extracts were prepared. Lysates were immunoblotted with antibodies to V5, LDH, LaminB, Cul3 and Rbx1.
Figure Legend Snippet: Subcellular localization of mutant forms of INrf2. ( A – D ) HepG2 cells were transfected with 1 μg of mutant plasmids encoding INrf2Y85A, INrf2Y141A, INrf2Y208A or INrf2Y255A. Cells were then treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were harvested and nuclear and cytosolic extracts were prepared. Lysates were immunoblotted with antibodies to V5, LDH, LaminB, Cul3 and Rbx1.

Techniques Used: Mutagenesis, Transfection

Cytosolic accumulation and degradation of the INrf2–Cul3–Rbx1 complex. ( A – C ) HepG2 cells were pre-treated with 2 μM MG132 for 16 hours. HepG2 cells were then treated with 100 μM t -BHQ and MG132 for the indicated periods of time. Cells were harvested, and nuclear and cytosolic extracts were prepared. Lysates were immunoblotted. The densitometry measurements of bands were quantified and relative intensities are presented below the blots by numbers. (A) Endogenous INrf2, Cul3, Rbx1, LDH and LaminB were probed. (B) 1 μg of vector encoding INrf2–V5 was transiently transfected; INrf2–V5, Cul3, Rbx1, LDH and lamin B were probed. (C) 1 μg of vector encoding the INrf2Y85A–V5 mutant was transiently transfected; INrf2Y85A–V5, Cul3, Rbx1, LDH and lamin B were probed. ( D – F ) HepG2 cells were transfected with vector encoding INrf2–V5, Cul3–V5 or Myc–Rbx1 and HA–Ub and pre-treated with 2 μM MG132 for 16 hours. Cells were then treated with 100 μM t -BHQ and MG132 for the indicated periods of time. Protein was aliquoted from samples and used for inputs. The rest of the sample (1 mg) was immunoprecipitated with antibodies to V5 or myc and immunoblotted with antibodies to HA.
Figure Legend Snippet: Cytosolic accumulation and degradation of the INrf2–Cul3–Rbx1 complex. ( A – C ) HepG2 cells were pre-treated with 2 μM MG132 for 16 hours. HepG2 cells were then treated with 100 μM t -BHQ and MG132 for the indicated periods of time. Cells were harvested, and nuclear and cytosolic extracts were prepared. Lysates were immunoblotted. The densitometry measurements of bands were quantified and relative intensities are presented below the blots by numbers. (A) Endogenous INrf2, Cul3, Rbx1, LDH and LaminB were probed. (B) 1 μg of vector encoding INrf2–V5 was transiently transfected; INrf2–V5, Cul3, Rbx1, LDH and lamin B were probed. (C) 1 μg of vector encoding the INrf2Y85A–V5 mutant was transiently transfected; INrf2Y85A–V5, Cul3, Rbx1, LDH and lamin B were probed. ( D – F ) HepG2 cells were transfected with vector encoding INrf2–V5, Cul3–V5 or Myc–Rbx1 and HA–Ub and pre-treated with 2 μM MG132 for 16 hours. Cells were then treated with 100 μM t -BHQ and MG132 for the indicated periods of time. Protein was aliquoted from samples and used for inputs. The rest of the sample (1 mg) was immunoprecipitated with antibodies to V5 or myc and immunoblotted with antibodies to HA.

Techniques Used: Plasmid Preparation, Transfection, Mutagenesis, Immunoprecipitation

Nuclear accumulation of INrf2Y85A shows no effect on Nrf2 activation but interferes with nuclear removal of Nrf2. ( A ) Nrf2 ubiquitylation. HepG2 cells were co-transfected with constructs encoding FLAG–Nrf2, INrf2–V5 or mutant INrf2Y85A–V5 and HA–Ub for 24 hours. Cells were pretreated with MG132 (5 μM) for 2 hours and post-treated with DMSO or t -BHQ (100 μM) for the indicated periods of time. Cells were harvested and nuclear and cytosolic extracts prepared by Active Motif Kit. 500 μg of nuclear and cytosolic extracts were immunoprecipitated with antibody against FLAG and western blotted for HA–HRP antibody (left top panels). Nuclear and cytosolic ubiquitylation of FLAG–Nrf2 was measured by using Quantity-One 4.6.3 Image software (ChemiDoc XRS, Bio-Rad) and plotted (right top panels). 60 μg of nuclear lysates from the above were also immunoblotted with antibodies against FLAG, V5 and lamin-B (left bottom panel). After immunoblotting, the intensities of protein bands of nuclear FLAG–Nrf2, INrf2–V5 and mutant INrf2Y85A–V5 were measured by using Quantity-One 4.6.3. Image software, normalized against proper loading controls and plotted (right bottom panel). ( B ) The INrf2Y85A mutant stabilized Nrf2. HepG2 cells were transfected wild-type INrf2–V5 or mutant INrf2Y85A–V5 for 24 hours. Cells were treated with DMSO or t -BHQ (100 μM) for the indicated periods of time. Cells were harvested and 60 μg whole-cell lysates were immunoblotted with antibodies to Nrf2, actin and NQO1 (upper panel). After immunoblotting, the intensities of NQO1 protein bands were measured and plotted (lower panel). ( C ) Luciferase assay: HepG2 cells were grown in monolayer cultures in 12-well plates for 12 hours and co-transfected with vector encoding INrf2–V5 or mutant INrf2Y85A–V5 (100 ng well −1 ) along with 100 ng well −1 of NQO1 promoter ARE–Luc reporter construct and 10 ng well −1 of firefly Renilla luciferase plasmid pRL-TK. After 24 hours of transfection, cells were treated with DMSO or t -BHQ (100 μM) for 4 to 16 hours. NQO1 promoter luciferase activity was measured and plotted as described in the Materials and Methods. The data are shown as the means ± s.d. of three independent transfection experiments.
Figure Legend Snippet: Nuclear accumulation of INrf2Y85A shows no effect on Nrf2 activation but interferes with nuclear removal of Nrf2. ( A ) Nrf2 ubiquitylation. HepG2 cells were co-transfected with constructs encoding FLAG–Nrf2, INrf2–V5 or mutant INrf2Y85A–V5 and HA–Ub for 24 hours. Cells were pretreated with MG132 (5 μM) for 2 hours and post-treated with DMSO or t -BHQ (100 μM) for the indicated periods of time. Cells were harvested and nuclear and cytosolic extracts prepared by Active Motif Kit. 500 μg of nuclear and cytosolic extracts were immunoprecipitated with antibody against FLAG and western blotted for HA–HRP antibody (left top panels). Nuclear and cytosolic ubiquitylation of FLAG–Nrf2 was measured by using Quantity-One 4.6.3 Image software (ChemiDoc XRS, Bio-Rad) and plotted (right top panels). 60 μg of nuclear lysates from the above were also immunoblotted with antibodies against FLAG, V5 and lamin-B (left bottom panel). After immunoblotting, the intensities of protein bands of nuclear FLAG–Nrf2, INrf2–V5 and mutant INrf2Y85A–V5 were measured by using Quantity-One 4.6.3. Image software, normalized against proper loading controls and plotted (right bottom panel). ( B ) The INrf2Y85A mutant stabilized Nrf2. HepG2 cells were transfected wild-type INrf2–V5 or mutant INrf2Y85A–V5 for 24 hours. Cells were treated with DMSO or t -BHQ (100 μM) for the indicated periods of time. Cells were harvested and 60 μg whole-cell lysates were immunoblotted with antibodies to Nrf2, actin and NQO1 (upper panel). After immunoblotting, the intensities of NQO1 protein bands were measured and plotted (lower panel). ( C ) Luciferase assay: HepG2 cells were grown in monolayer cultures in 12-well plates for 12 hours and co-transfected with vector encoding INrf2–V5 or mutant INrf2Y85A–V5 (100 ng well −1 ) along with 100 ng well −1 of NQO1 promoter ARE–Luc reporter construct and 10 ng well −1 of firefly Renilla luciferase plasmid pRL-TK. After 24 hours of transfection, cells were treated with DMSO or t -BHQ (100 μM) for 4 to 16 hours. NQO1 promoter luciferase activity was measured and plotted as described in the Materials and Methods. The data are shown as the means ± s.d. of three independent transfection experiments.

Techniques Used: Activation Assay, Transfection, Construct, Mutagenesis, Immunoprecipitation, Western Blot, Software, Luciferase, Plasmid Preparation, Activity Assay

Subcellular localization of endogenous and overexpressed INrf2. HepG2 cells were treated with chemicals for various periods of time and then harvested and nuclear and cytosolic extracts prepared. Lysates were immunoblotted. Densitometry measurements of bands were quantified and their values are shown below, relative to control DMSO measurements. Anti-LDH (cytosolic control) and anti-lamin B (nuclear control) were probed in all blots. ( A ) Cells were treated with vehicle control (DMSO) or 100 μM t -BHQ. Endogenous INrf2, Cul3 and Rbx1 were probed. ( B ) HepG2 cells were transfected with 1 μg of INrf2–V5 plasmid for 24 hours, and cells were treated with DMSO or 100 μM t -BHQ, as indicated. The lysates were immunoblotted with antibodies against V5, Cul3 and Rbx1. ( C ) Cells were pre-treated with 100 μM genistein for 2 hours, and the cells were then treated with either DMSO or 100 μM t -BHQ along with genistein for the indicated periods of time. Lysates were immunoblotted with antibodies to INrf2, Cul3 and Rbx1. ( D ) Cells were treated with 20 ng ml −1 of LMB for 2 hours; cells were then treated with either DMSO or 100 μM t -BHQ along with LMB for the indicated periods of time. Lysates were immunoblotted with antibodies to INrf2, Cul3 and Rbx1.
Figure Legend Snippet: Subcellular localization of endogenous and overexpressed INrf2. HepG2 cells were treated with chemicals for various periods of time and then harvested and nuclear and cytosolic extracts prepared. Lysates were immunoblotted. Densitometry measurements of bands were quantified and their values are shown below, relative to control DMSO measurements. Anti-LDH (cytosolic control) and anti-lamin B (nuclear control) were probed in all blots. ( A ) Cells were treated with vehicle control (DMSO) or 100 μM t -BHQ. Endogenous INrf2, Cul3 and Rbx1 were probed. ( B ) HepG2 cells were transfected with 1 μg of INrf2–V5 plasmid for 24 hours, and cells were treated with DMSO or 100 μM t -BHQ, as indicated. The lysates were immunoblotted with antibodies against V5, Cul3 and Rbx1. ( C ) Cells were pre-treated with 100 μM genistein for 2 hours, and the cells were then treated with either DMSO or 100 μM t -BHQ along with genistein for the indicated periods of time. Lysates were immunoblotted with antibodies to INrf2, Cul3 and Rbx1. ( D ) Cells were treated with 20 ng ml −1 of LMB for 2 hours; cells were then treated with either DMSO or 100 μM t -BHQ along with LMB for the indicated periods of time. Lysates were immunoblotted with antibodies to INrf2, Cul3 and Rbx1.

Techniques Used: Transfection, Plasmid Preparation

Immunoprecipitation of INrf2, INrf2–V5 and INrf2Y85A–V5. ( A ) HepG2 cells were treated with either DMSO or 100 μM t -BHQ for immunoprecipitations. Cells were then collected, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody against INrf2 and western blotted with antibodies to phosphotyrosine and INrf2 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibodies to INrf2 and phosphotyrosine (bottom panels). ( B , C ) HepG2 cells were transfected with 1 μg of INrf2–V5 or INrf2Y85A–V5 and treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were harvested, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to phosphotyrosine and V5 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibody to V5 (bottom panels).
Figure Legend Snippet: Immunoprecipitation of INrf2, INrf2–V5 and INrf2Y85A–V5. ( A ) HepG2 cells were treated with either DMSO or 100 μM t -BHQ for immunoprecipitations. Cells were then collected, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody against INrf2 and western blotted with antibodies to phosphotyrosine and INrf2 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibodies to INrf2 and phosphotyrosine (bottom panels). ( B , C ) HepG2 cells were transfected with 1 μg of INrf2–V5 or INrf2Y85A–V5 and treated with either DMSO or 100 μM t -BHQ for the indicated periods of time. Cells were harvested, and nuclear and cytosolic fractions were separated. 1 mg of nuclear lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to phosphotyrosine and V5 (top panels). 1 mg of nuclear lysate was immunoprecipitated with antibody to phosphotyrosine and western blotted with antibody to V5 (bottom panels).

Techniques Used: Immunoprecipitation, Western Blot, Transfection

INrf2Y85A is able to bind to Cul3–Rbx1 and Nrf2. ( A , B ) HepG2 cells were transfected with 1 μg vector encoding (A) INrf2–V5 or (B) INrf2Y85A–V5 and 500 ng vector encoding HA–Cul3 or Myc–Rbx1. Cells were then treated with 100 μM t -BHQ for the indicated periods of time. Cells were harvested; 1 mg of lysates were immunoprecipitated with antibodies against V5, HA or Myc and western blotted with antibodies against V5, HA or Myc. ( C , D ) HepG2 cells were transfected with 1 μg vector encoding (C) INrf2–V5 or (D) INrf2Y85A–V5 and pretreated with 2 μM MG132 for 16 hours. Cells were then treated with 100 μM t -BHQ for the indicated periods of time. 1 mg of lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to Nrf2 and V5 (top panels). 1 mg of lysate was immunoprecipitated with antibody to Nrf2 and western blotted with antibodies to V5 and Nrf2 (bottom panels).
Figure Legend Snippet: INrf2Y85A is able to bind to Cul3–Rbx1 and Nrf2. ( A , B ) HepG2 cells were transfected with 1 μg vector encoding (A) INrf2–V5 or (B) INrf2Y85A–V5 and 500 ng vector encoding HA–Cul3 or Myc–Rbx1. Cells were then treated with 100 μM t -BHQ for the indicated periods of time. Cells were harvested; 1 mg of lysates were immunoprecipitated with antibodies against V5, HA or Myc and western blotted with antibodies against V5, HA or Myc. ( C , D ) HepG2 cells were transfected with 1 μg vector encoding (C) INrf2–V5 or (D) INrf2Y85A–V5 and pretreated with 2 μM MG132 for 16 hours. Cells were then treated with 100 μM t -BHQ for the indicated periods of time. 1 mg of lysate was immunoprecipitated with antibody to V5 and western blotted with antibodies to Nrf2 and V5 (top panels). 1 mg of lysate was immunoprecipitated with antibody to Nrf2 and western blotted with antibodies to V5 and Nrf2 (bottom panels).

Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

2) Product Images from "Antioxidant-induced modification of INrf2 cysteine 151 and PKC-?-mediated phosphorylation of Nrf2 serine 40 are both required for stabilization and nuclear translocation of Nrf2 and increased drug resistance"

Article Title: Antioxidant-induced modification of INrf2 cysteine 151 and PKC-?-mediated phosphorylation of Nrf2 serine 40 are both required for stabilization and nuclear translocation of Nrf2 and increased drug resistance

Journal: Journal of Cell Science

doi: 10.1242/jcs.058537

Cysteine 151 of INrf2 is required for releasing Nrf2 from INrf2 in response to antioxidant. (A,B) Western blot analysis (A) and immunoprecipitation (B). Hepa-1 cells were co-transfected with FLAG-Nrf2, INrf2-V5 or mutant INrf2C151A-V5 and another set
Figure Legend Snippet: Cysteine 151 of INrf2 is required for releasing Nrf2 from INrf2 in response to antioxidant. (A,B) Western blot analysis (A) and immunoprecipitation (B). Hepa-1 cells were co-transfected with FLAG-Nrf2, INrf2-V5 or mutant INrf2C151A-V5 and another set

Techniques Used: Western Blot, Immunoprecipitation, Transfection, Mutagenesis

tBHQ induces destabilization of INrf2 and triggers ubiquitylation and degradation of INrf2 in PKC-δ efficient and deficient cells. (A) Western blot analysis of degradation of INrf2 and mutant INrf2C151A proteins. Hepa-1 cells were transfected
Figure Legend Snippet: tBHQ induces destabilization of INrf2 and triggers ubiquitylation and degradation of INrf2 in PKC-δ efficient and deficient cells. (A) Western blot analysis of degradation of INrf2 and mutant INrf2C151A proteins. Hepa-1 cells were transfected

Techniques Used: Western Blot, Mutagenesis, Transfection

Antioxidant-induced PKC-δ-mediated phosphorylation of Nrf2S40 led to dissociation of Nrf2 from INrf2. (A) Western blot analysis. Hepa-1, PKC-δ +/+ and PKC-δ –/– cells were treated with DMSO or tBHQ (50 mM) for
Figure Legend Snippet: Antioxidant-induced PKC-δ-mediated phosphorylation of Nrf2S40 led to dissociation of Nrf2 from INrf2. (A) Western blot analysis. Hepa-1, PKC-δ +/+ and PKC-δ –/– cells were treated with DMSO or tBHQ (50 mM) for

Techniques Used: Western Blot

A model showing antioxidant-mediated oxidation and destabilization of INrf2 followed by PKC-δ phosphorylation of Nrf2S40 leading to separation, stabilization and nuclear localization of Nrf2 and activation of ARE-gene expression and drug resistance.
Figure Legend Snippet: A model showing antioxidant-mediated oxidation and destabilization of INrf2 followed by PKC-δ phosphorylation of Nrf2S40 leading to separation, stabilization and nuclear localization of Nrf2 and activation of ARE-gene expression and drug resistance.

Techniques Used: Activation Assay, Expressing

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Blocking Assay:

Article Title: Protective Effects of Curcumin on Renal Oxidative Stress and Lipid Metabolism in a Rat Model of Type 2 Diabetic Nephropathy
Article Snippet: .. The membrane was hybridized with anti-p-AMPK, anti-AMPK, and anti-p-ACC antibodies (Cell Signaling Technology Inc., Danvers, MA, USA), or anti-β-actin, anti-Nrf2, anti-Keap1, anti-HO-1, anti-vascular endothelial growth factor, anti-tumor growth factor-β1, anti-SREBP1, anti-SREBP2, and anti-adipose differentiation related protein (ADRP) antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in a blocking buffer overnight at 4℃. .. The filter was incubated with anti-rabbit or anti-mouse for 60 minutes at room temperature.

Enzyme-linked Immunosorbent Assay:

Article Title: Imidazoline I2 receptor inhibitor idazoxan regulates the progression of hepatic fibrosis via Akt-Nrf2-Smad2/3 signaling pathway
Article Snippet: Anti-NQO1, anti-Smad2, anti-phospho-Smad2, anti-Smad3, anti-p-Smad3, anti-keap1, anti-GAPDH, anti-PCNA, anti-SOD2, anti-Catalase, anti-p38, anti-p-p38, anti-Akt and anti-p-Akt were obtained from Santa Cruz (CA, USA). .. IL-1β, IL-6, TNF-α and TGF-β enzyme-linked immunosorbent assay (ELISA) kits were obtained from Boster Biotechnology (Wuhan, China).

Incubation:

Article Title: Protective Effects of Curcumin on Renal Oxidative Stress and Lipid Metabolism in a Rat Model of Type 2 Diabetic Nephropathy
Article Snippet: The membrane was hybridized with anti-p-AMPK, anti-AMPK, and anti-p-ACC antibodies (Cell Signaling Technology Inc., Danvers, MA, USA), or anti-β-actin, anti-Nrf2, anti-Keap1, anti-HO-1, anti-vascular endothelial growth factor, anti-tumor growth factor-β1, anti-SREBP1, anti-SREBP2, and anti-adipose differentiation related protein (ADRP) antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in a blocking buffer overnight at 4℃. .. The filter was incubated with anti-rabbit or anti-mouse for 60 minutes at room temperature.

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).

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: 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.

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: Protective Effects of Curcumin on Renal Oxidative Stress and Lipid Metabolism in a Rat Model of Type 2 Diabetic Nephropathy
Article Snippet: Paragraph title: Western blot analysis in renal cortex tissue ... The membrane was hybridized with anti-p-AMPK, anti-AMPK, and anti-p-ACC antibodies (Cell Signaling Technology Inc., Danvers, MA, USA), or anti-β-actin, anti-Nrf2, anti-Keap1, anti-HO-1, anti-vascular endothelial growth factor, anti-tumor growth factor-β1, anti-SREBP1, anti-SREBP2, and anti-adipose differentiation related protein (ADRP) antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in a blocking buffer overnight at 4℃.

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: 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).

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).

Transfection:

Article Title: Direct interaction between Nrf2 and p21Cip1/WAF1 upregulates the Nrf2-mediated antioxidant response
Article Snippet: Paragraph title: Antibodies, Transfection, Animal Treatment, Immunoblot Analysis, and Reporter Gene Assay ... Anti-Nrf2, anti-Keap1, and anti-p21 (Santa Cruz); anti-HA beads (Sigma); anti-HA (Covance); Chitin beads and the anti-CBD (New England Biolab) were purchased from commercial sources.

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: 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.

Reporter Gene Assay:

Article Title: Direct interaction between Nrf2 and p21Cip1/WAF1 upregulates the Nrf2-mediated antioxidant response
Article Snippet: Paragraph title: Antibodies, Transfection, Animal Treatment, Immunoblot Analysis, and Reporter Gene Assay ... Anti-Nrf2, anti-Keap1, and anti-p21 (Santa Cruz); anti-HA beads (Sigma); anti-HA (Covance); Chitin beads and the anti-CBD (New England Biolab) were purchased from commercial sources.

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).

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).

Protein Extraction:

Article Title: Protective Effects of Curcumin on Renal Oxidative Stress and Lipid Metabolism in a Rat Model of Type 2 Diabetic Nephropathy
Article Snippet: Western blot analysis in renal cortex tissue Renal cortex protein was extracted using PRO-PREPTM protein Extraction Solution (Intron Biotechnology, Seoul, Korea) by Tissue Lyser II (QIAGEN GmbH, Haan, Germany). .. The membrane was hybridized with anti-p-AMPK, anti-AMPK, and anti-p-ACC antibodies (Cell Signaling Technology Inc., Danvers, MA, USA), or anti-β-actin, anti-Nrf2, anti-Keap1, anti-HO-1, anti-vascular endothelial growth factor, anti-tumor growth factor-β1, anti-SREBP1, anti-SREBP2, and anti-adipose differentiation related protein (ADRP) antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in a blocking buffer overnight at 4℃.

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: 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).

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).

Electrochemiluminescence:

Article Title: Protective Effects of Curcumin on Renal Oxidative Stress and Lipid Metabolism in a Rat Model of Type 2 Diabetic Nephropathy
Article Snippet: The membrane was hybridized with anti-p-AMPK, anti-AMPK, and anti-p-ACC antibodies (Cell Signaling Technology Inc., Danvers, MA, USA), or anti-β-actin, anti-Nrf2, anti-Keap1, anti-HO-1, anti-vascular endothelial growth factor, anti-tumor growth factor-β1, anti-SREBP1, anti-SREBP2, and anti-adipose differentiation related protein (ADRP) antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in a blocking buffer overnight at 4℃. .. Specific signals were detected using the electrochemiluminescence solution (Santa Cruz Biotechnology Inc.).

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    Santa Cruz Biotechnology antibodies against keap1
    The interaction between p62 and <t>Keap1</t> and the domains that are required for the interaction. (A) Identification of the Keap1-interacting protein p62. Two stable cell lines, MDA-MB-231 and Keap1 −/− , that expressed either vector control
    Antibodies Against Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The interaction between p62 and Keap1 and the domains that are required for the interaction. (A) Identification of the Keap1-interacting protein p62. Two stable cell lines, MDA-MB-231 and Keap1 −/− , that expressed either vector control

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: The interaction between p62 and Keap1 and the domains that are required for the interaction. (A) Identification of the Keap1-interacting protein p62. Two stable cell lines, MDA-MB-231 and Keap1 −/− , that expressed either vector control

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques: Stable Transfection, Multiple Displacement Amplification, Plasmid Preparation

    p62 decreased ubiquitination of Nrf2, leading to an increase in Nrf2 stability. (A) p62 decreased the ubiquitination of Nrf2 and increased the ubiquitination of Keap1. HEK293 cells were cotransfected with an expression vector for Nrf2, Keap1, HA-ubiquitin,

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: p62 decreased ubiquitination of Nrf2, leading to an increase in Nrf2 stability. (A) p62 decreased the ubiquitination of Nrf2 and increased the ubiquitination of Keap1. HEK293 cells were cotransfected with an expression vector for Nrf2, Keap1, HA-ubiquitin,

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques: Expressing, Plasmid Preparation

    p62 sequestered Keap1 into aggregates. (A) The cellular localization of p62, Keap1, Nrf2, and Cul3. HEK293 cells were singly transfected with an expression vector for the fluorescently tagged protein. The subcellular localization of the proteins was monitored

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: p62 sequestered Keap1 into aggregates. (A) The cellular localization of p62, Keap1, Nrf2, and Cul3. HEK293 cells were singly transfected with an expression vector for the fluorescently tagged protein. The subcellular localization of the proteins was monitored

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques: Transfection, Expressing, Plasmid Preparation

    Autophagy-defective cells sequestered Keap1 into aggregates. (A and B) Keap1 was sequestered into aggregates in primary autophagy-deficient cells. Atg5 +/+ , Atg5 −/− , Beclin1 +/+ , and Beclin +/−

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: Autophagy-defective cells sequestered Keap1 into aggregates. (A and B) Keap1 was sequestered into aggregates in primary autophagy-deficient cells. Atg5 +/+ , Atg5 −/− , Beclin1 +/+ , and Beclin +/−

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques:

    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

    Journal: Neuroimmunomodulation

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

    doi: 10.1159/000347161

    Figure Lengend 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

    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).

    Techniques: 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

    Journal: Neuroimmunomodulation

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

    doi: 10.1159/000347161

    Figure Lengend 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

    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).

    Techniques: Injection, Western Blot

    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