Structured Review

Cell Signaling Technology Inc anti nqo1
Effect of KEAP1 knockdown on the expression of NRF2 and downstream targets. (A) mRNA expression levels of NRF2, <t>NQO1</t> and HO1 in Hep2, scHep2 and shKEAP1 Hep2 cells. Expression levels of NRF2, NQO1 and HO1 were increased following the knockdown of KEAP1 in Hep2 cells. (B) Representative NRF2 immunofluorescence staining images indicate that NRF2 translocated into the nuclei from the cytoplasm following knockdown of KEAP1 in Hep2 cells (magnification, ×40). (C) Western blotting demonstrated that nuclear NRF2 protein expression levels were elevated, while cytoplasmic NRF2 protein expression levels were reduced, following the knockdown of KEAP1 in Hep2 cells. (D) Western blotting demonstrated that total NQO1 and HO1 protein expression levels were increased within shKEAP1 Hep2 cells, compared with the scHep2 group. *P
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1) Product Images from "Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells"

Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2018.9288

Effect of KEAP1 knockdown on the expression of NRF2 and downstream targets. (A) mRNA expression levels of NRF2, NQO1 and HO1 in Hep2, scHep2 and shKEAP1 Hep2 cells. Expression levels of NRF2, NQO1 and HO1 were increased following the knockdown of KEAP1 in Hep2 cells. (B) Representative NRF2 immunofluorescence staining images indicate that NRF2 translocated into the nuclei from the cytoplasm following knockdown of KEAP1 in Hep2 cells (magnification, ×40). (C) Western blotting demonstrated that nuclear NRF2 protein expression levels were elevated, while cytoplasmic NRF2 protein expression levels were reduced, following the knockdown of KEAP1 in Hep2 cells. (D) Western blotting demonstrated that total NQO1 and HO1 protein expression levels were increased within shKEAP1 Hep2 cells, compared with the scHep2 group. *P
Figure Legend Snippet: Effect of KEAP1 knockdown on the expression of NRF2 and downstream targets. (A) mRNA expression levels of NRF2, NQO1 and HO1 in Hep2, scHep2 and shKEAP1 Hep2 cells. Expression levels of NRF2, NQO1 and HO1 were increased following the knockdown of KEAP1 in Hep2 cells. (B) Representative NRF2 immunofluorescence staining images indicate that NRF2 translocated into the nuclei from the cytoplasm following knockdown of KEAP1 in Hep2 cells (magnification, ×40). (C) Western blotting demonstrated that nuclear NRF2 protein expression levels were elevated, while cytoplasmic NRF2 protein expression levels were reduced, following the knockdown of KEAP1 in Hep2 cells. (D) Western blotting demonstrated that total NQO1 and HO1 protein expression levels were increased within shKEAP1 Hep2 cells, compared with the scHep2 group. *P

Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot

Effect of KEAP1 knockdown on H 2 O 2 -induced alterations in the expression of NQO1 and HO1. mRNA levels of (A) NQO1 and (B) HO1 within shKEAP1 Hep2 cells were increased in a dose- and time-dependent manner following exposure to H 2 O 2 . KEAP1 knockdown upregulated the expression levels of NQO1 and HO1 compared with in scHep2 cells. *P
Figure Legend Snippet: Effect of KEAP1 knockdown on H 2 O 2 -induced alterations in the expression of NQO1 and HO1. mRNA levels of (A) NQO1 and (B) HO1 within shKEAP1 Hep2 cells were increased in a dose- and time-dependent manner following exposure to H 2 O 2 . KEAP1 knockdown upregulated the expression levels of NQO1 and HO1 compared with in scHep2 cells. *P

Techniques Used: Expressing

2) Product Images from "The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2) activation"

Article Title: The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2) activation

Journal: Redox Biology

doi: 10.1016/j.redox.2015.12.005

Effects of RTA 408 on expression of Nrf2 and downstream genes . (A) Dose-dependent effects of RTA 408 on Nrf2 and downstream gene expression. RPE cells were pretreated with 1–100 nM RTA for 24 h. Control cells were treated identically, but without addition of RTA 408. Whole cell lysates of each group were prepared, and 60 ug of each protein sample was used to detect the levels of Nrf2, SOD2, catalase, NQO1, HO-1, Grx1, and Trx1 by western immunoblotting. GAPDH was used as a loading control. (B) Time-dependent effects of RTA 408 on Nrf2 and its downstream gene expression. Cells were treated with 100 nM RTA for various time (0–24 h). 60 μg of each protein sample was subjected to western immunoblot analysis. The blot was incubated with GAPDH, Nrf2, SOD2, catalase, NQO1, HO-1, Grx1, and Trx1 antibodies. GAPDH was used as a reference for equal protein loading. All experiments were repeated three times with similar results. (C) Nrf2 expression in the cytoplasmic and nuclear fractions. Cells were treated with 100 nM of RTA 408 for various time points (0, 0.5, 2 and 6 h) and then were separated to its nuclear and cytoplasmic portions using a kit. 40 μg of each sample were subjected to western immunoblot analysis and incubated with B23, Nrf2, and β-actin antibodies. All experiments were repeated three times with similar results. (D) Quantitative analysis of time-dependent Nrf2 translocation are depicted as mean±SD, ** P
Figure Legend Snippet: Effects of RTA 408 on expression of Nrf2 and downstream genes . (A) Dose-dependent effects of RTA 408 on Nrf2 and downstream gene expression. RPE cells were pretreated with 1–100 nM RTA for 24 h. Control cells were treated identically, but without addition of RTA 408. Whole cell lysates of each group were prepared, and 60 ug of each protein sample was used to detect the levels of Nrf2, SOD2, catalase, NQO1, HO-1, Grx1, and Trx1 by western immunoblotting. GAPDH was used as a loading control. (B) Time-dependent effects of RTA 408 on Nrf2 and its downstream gene expression. Cells were treated with 100 nM RTA for various time (0–24 h). 60 μg of each protein sample was subjected to western immunoblot analysis. The blot was incubated with GAPDH, Nrf2, SOD2, catalase, NQO1, HO-1, Grx1, and Trx1 antibodies. GAPDH was used as a reference for equal protein loading. All experiments were repeated three times with similar results. (C) Nrf2 expression in the cytoplasmic and nuclear fractions. Cells were treated with 100 nM of RTA 408 for various time points (0, 0.5, 2 and 6 h) and then were separated to its nuclear and cytoplasmic portions using a kit. 40 μg of each sample were subjected to western immunoblot analysis and incubated with B23, Nrf2, and β-actin antibodies. All experiments were repeated three times with similar results. (D) Quantitative analysis of time-dependent Nrf2 translocation are depicted as mean±SD, ** P

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

Effects of RTA 408 on Grx1, Trx1, and NQO1 enzyme activities . RPE cells were pretreated with 100 nM RTA 408 for 24 h followed by 200 μM H 2 O 2 for another 6 h. Quantitative analysis of Grx1 (A), Trx1 (B), and (C) NQO1 activity of all treatment groups (control, H 2 O 2 -treated, RTA 408 and H 2 O 2 -treated, and 100 nM RTA 408 only RPE cells) are depicted as mean±SD, ** P
Figure Legend Snippet: Effects of RTA 408 on Grx1, Trx1, and NQO1 enzyme activities . RPE cells were pretreated with 100 nM RTA 408 for 24 h followed by 200 μM H 2 O 2 for another 6 h. Quantitative analysis of Grx1 (A), Trx1 (B), and (C) NQO1 activity of all treatment groups (control, H 2 O 2 -treated, RTA 408 and H 2 O 2 -treated, and 100 nM RTA 408 only RPE cells) are depicted as mean±SD, ** P

Techniques Used: Activity Assay

RTA 408 activates the Nrf2 pathway which leads to an upregulation of antioxidant enzymes and protects the cell from oxidative stress . Binding of RTA 408 to Cys151 in Keap1, the negative regulator of Nrf2, results in Keap1 inhibition. This promotes Nrf2 movement into the nucleus where it binds to the antioxidant response element (ARE). With the activation of ARE, transcriptional activation of antioxidant enzymes heme oxygenase-1 (HO-1), NADPH dehydrogenase (NQO1), superoxide dismutase 2 (SOD2), catalase, thioredoxin 1 (Trx1), and glutaredoxin 1 (Grx1) occurs. HO-1 converts heme to carbon monoxide, iron (II), and biliverdin, which all indirectly scavenge ROS. NQO1 converts enzymes and other proteins (R) back to their reduced form (RH) through the electron transfer between NADPH and NADP. Superoxide (O 2 − ) can be converted to hydrogen peroxide using SOD2 and then further processed into water by catalase. Grx1 and Trx1 work together to reduce protein-glutathione mixed disulfide (PSSG) and protein-protein disulfide (PSSP) to protect protein thiols from oxidation. Overall, RTA 408 induction of phase II antioxidant enzymes such as HO-1, NQO1, SOD2, catalase, Grx1, and Trx1 via activation of Nrf2 promote RPE cell survival during oxidative stress.
Figure Legend Snippet: RTA 408 activates the Nrf2 pathway which leads to an upregulation of antioxidant enzymes and protects the cell from oxidative stress . Binding of RTA 408 to Cys151 in Keap1, the negative regulator of Nrf2, results in Keap1 inhibition. This promotes Nrf2 movement into the nucleus where it binds to the antioxidant response element (ARE). With the activation of ARE, transcriptional activation of antioxidant enzymes heme oxygenase-1 (HO-1), NADPH dehydrogenase (NQO1), superoxide dismutase 2 (SOD2), catalase, thioredoxin 1 (Trx1), and glutaredoxin 1 (Grx1) occurs. HO-1 converts heme to carbon monoxide, iron (II), and biliverdin, which all indirectly scavenge ROS. NQO1 converts enzymes and other proteins (R) back to their reduced form (RH) through the electron transfer between NADPH and NADP. Superoxide (O 2 − ) can be converted to hydrogen peroxide using SOD2 and then further processed into water by catalase. Grx1 and Trx1 work together to reduce protein-glutathione mixed disulfide (PSSG) and protein-protein disulfide (PSSP) to protect protein thiols from oxidation. Overall, RTA 408 induction of phase II antioxidant enzymes such as HO-1, NQO1, SOD2, catalase, Grx1, and Trx1 via activation of Nrf2 promote RPE cell survival during oxidative stress.

Techniques Used: Binding Assay, Inhibition, Activation Assay

3) Product Images from "Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2"

Article Title: Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005581

SFN does not trigger expression of interferon-stimulated anti-viral factors SAMHD1 or MX2. PMA-differentiated THP1 cells were mock-treated or treated with media supplemented with vehicle only (DMSO) or with 10 μM SFN or with 500 U/mL of IFNα. (A), Proteins from whole cell lysates were resolved by SDS-PAGE and identified by western blotting using antibodies with the indicated specificities. (B), Densitometric analysis was performed on the Nrf2, SAMHD1, MX2, NQO1 and GCLM (NQO1 and GCLM are both indicators of Nrf2 function) bands and normalized to the values of the corresponding tubulin bands. The relative normalized intensities of the bands were then graphed.
Figure Legend Snippet: SFN does not trigger expression of interferon-stimulated anti-viral factors SAMHD1 or MX2. PMA-differentiated THP1 cells were mock-treated or treated with media supplemented with vehicle only (DMSO) or with 10 μM SFN or with 500 U/mL of IFNα. (A), Proteins from whole cell lysates were resolved by SDS-PAGE and identified by western blotting using antibodies with the indicated specificities. (B), Densitometric analysis was performed on the Nrf2, SAMHD1, MX2, NQO1 and GCLM (NQO1 and GCLM are both indicators of Nrf2 function) bands and normalized to the values of the corresponding tubulin bands. The relative normalized intensities of the bands were then graphed.

Techniques Used: Expressing, SDS Page, Western Blot

SFN acts through Nrf2 to block HIV infection in macrophages. PMA-differentiated THP1 cells were pretreated for twenty-four hours with the Nrf2 activators: (A), SFN, (B), DMF, or (C), EGCG at the indicated concentrations. Pretreatment of cultures with 5 μM of the reverse transcription inhibitor zidovudine (AZT) served as a positive control for viral inhibition. Twenty-four hours after treatment, cells were either mock infected or infected with VSV-G-pseudotyped HIV-1 encoding firefly luciferase in place of nef . Twenty-four hours after infection, the cells were harvested and luciferase activity was measured by photon emission. The bar graphs represent the data for replicate experiments (n = 3). (D), Cultures of hMDMs were transfected with either a non-targeting siRNA (control) or siRNA specific for Nrf2 mRNA. siRNA transfected hMDMs were either treated with vehicle (DMSO) or with 10 μM SFN. Twenty-four hours after treatment, the cells were either mock infected or infected with VSV-G-pseudotyped HIV-1 encoding firefly luciferase in place of nef . Twenty-four hours after infection, the cells were harvested and luciferase activity was measured by photon emission. The bar graphs represent the quantified data for replicate experiments (n = 3). (E) and (F), Representative samples from (D) were lysed and proteins from whole cell lysates were resolved by SDS-PAGE and identified by western blotting using antibodies with the indicated specificities. Densitometric analysis was performed on the Nrf2 and NQO1 (an indicator of Nrf2 function) bands and normalized to the values of the corresponding tubulin bands. The relative normalized intensities of the Nrf2 and NQO1 bands were then graphed. The data shown is representative of n = 3.
Figure Legend Snippet: SFN acts through Nrf2 to block HIV infection in macrophages. PMA-differentiated THP1 cells were pretreated for twenty-four hours with the Nrf2 activators: (A), SFN, (B), DMF, or (C), EGCG at the indicated concentrations. Pretreatment of cultures with 5 μM of the reverse transcription inhibitor zidovudine (AZT) served as a positive control for viral inhibition. Twenty-four hours after treatment, cells were either mock infected or infected with VSV-G-pseudotyped HIV-1 encoding firefly luciferase in place of nef . Twenty-four hours after infection, the cells were harvested and luciferase activity was measured by photon emission. The bar graphs represent the data for replicate experiments (n = 3). (D), Cultures of hMDMs were transfected with either a non-targeting siRNA (control) or siRNA specific for Nrf2 mRNA. siRNA transfected hMDMs were either treated with vehicle (DMSO) or with 10 μM SFN. Twenty-four hours after treatment, the cells were either mock infected or infected with VSV-G-pseudotyped HIV-1 encoding firefly luciferase in place of nef . Twenty-four hours after infection, the cells were harvested and luciferase activity was measured by photon emission. The bar graphs represent the quantified data for replicate experiments (n = 3). (E) and (F), Representative samples from (D) were lysed and proteins from whole cell lysates were resolved by SDS-PAGE and identified by western blotting using antibodies with the indicated specificities. Densitometric analysis was performed on the Nrf2 and NQO1 (an indicator of Nrf2 function) bands and normalized to the values of the corresponding tubulin bands. The relative normalized intensities of the Nrf2 and NQO1 bands were then graphed. The data shown is representative of n = 3.

Techniques Used: Blocking Assay, Infection, Positive Control, Inhibition, Luciferase, Activity Assay, Transfection, SDS Page, Western Blot

4) Product Images from "CO/HO-1 Induces NQO-1 Expression via Nrf2 Activation"

Article Title: CO/HO-1 Induces NQO-1 Expression via Nrf2 Activation

Journal: Immune Network

doi: 10.4110/in.2011.11.6.376

Effects of CORM-2 on Nrf2-mediated NQO1 promoter activity. HepG2 cells were co-transfected with pGL3/NQO1p-ARE and pcDNA3-Nrf2. After 24 h, cells were treated with 30 uM CORM-2 and at 6 h post-treatment, luciferase activity was determined. The levels of firefly luciferase activity were normalized to the Renilla luciferase activity. The relative luciferase activities are presented as a fold increase over no-treated cells. Each bar represents the mean±S.D. of three independent experiments ( * p
Figure Legend Snippet: Effects of CORM-2 on Nrf2-mediated NQO1 promoter activity. HepG2 cells were co-transfected with pGL3/NQO1p-ARE and pcDNA3-Nrf2. After 24 h, cells were treated with 30 uM CORM-2 and at 6 h post-treatment, luciferase activity was determined. The levels of firefly luciferase activity were normalized to the Renilla luciferase activity. The relative luciferase activities are presented as a fold increase over no-treated cells. Each bar represents the mean±S.D. of three independent experiments ( * p

Techniques Used: Activity Assay, Transfection, Luciferase

Effects of silencing of Nrf2 on CO-induced NQO1 promoter activity HepG2 cells were transfected with pGL3/NQO1p-501 containing the NQO1 promoter (-501 to +117), Nrf2 specific (siNRf2), and PERK specific (siPERK) or scrambled siRNA (scRNA). After 24 h cells were treated with various concentrations of CORM-2. At 6 h post-treatment, the level of firefly luciferase activity was normalized to the Renilla luciferase activity. The relative luciferase activities are presented as a fold increase over no-treated cells. Each bar represents the mean±S.D. of three independent experiments ( * p
Figure Legend Snippet: Effects of silencing of Nrf2 on CO-induced NQO1 promoter activity HepG2 cells were transfected with pGL3/NQO1p-501 containing the NQO1 promoter (-501 to +117), Nrf2 specific (siNRf2), and PERK specific (siPERK) or scrambled siRNA (scRNA). After 24 h cells were treated with various concentrations of CORM-2. At 6 h post-treatment, the level of firefly luciferase activity was normalized to the Renilla luciferase activity. The relative luciferase activities are presented as a fold increase over no-treated cells. Each bar represents the mean±S.D. of three independent experiments ( * p

Techniques Used: Activity Assay, Transfection, Luciferase

Time dependent NQO1 mRNA induction by exogenous CO. (A, B) HepG2 cells were treated with 50 uM CORM-2 or 250 ppm CO gas for indicated time points and analyzed. The mRNA expression of NQO1 was determined by semi-quantitative RT-PCR as described under Material and methods. GAPDH was used as internal controls.
Figure Legend Snippet: Time dependent NQO1 mRNA induction by exogenous CO. (A, B) HepG2 cells were treated with 50 uM CORM-2 or 250 ppm CO gas for indicated time points and analyzed. The mRNA expression of NQO1 was determined by semi-quantitative RT-PCR as described under Material and methods. GAPDH was used as internal controls.

Techniques Used: Expressing, Quantitative RT-PCR

Effects of exogenous CO on the NQO1 and HO-1 protein induction. HepG2 cells were treated with various concentrations of CORM-2 as exogenous CO donor for 6 h and the cell lysates were used for immunoblot with antibody against NQO1, HO-1, and β-actin.
Figure Legend Snippet: Effects of exogenous CO on the NQO1 and HO-1 protein induction. HepG2 cells were treated with various concentrations of CORM-2 as exogenous CO donor for 6 h and the cell lysates were used for immunoblot with antibody against NQO1, HO-1, and β-actin.

Techniques Used:

Effects of CORM-2 on the NQO1 promoter activity. HepG2 cells were transfected with hNQO1p-501 containing the NQO1 promoter (-501 to +117). After 24 h cells were treated with various concentrations of CORM-2. At 6 h post-treatment, the level of firefly luciferase activity was normalized to the Renilla luciferase activity. The relative luciferase activities are presented as a fold increase over no-treated cells. Each bar represents the mean±S.D. of three independent experiments ( ** p
Figure Legend Snippet: Effects of CORM-2 on the NQO1 promoter activity. HepG2 cells were transfected with hNQO1p-501 containing the NQO1 promoter (-501 to +117). After 24 h cells were treated with various concentrations of CORM-2. At 6 h post-treatment, the level of firefly luciferase activity was normalized to the Renilla luciferase activity. The relative luciferase activities are presented as a fold increase over no-treated cells. Each bar represents the mean±S.D. of three independent experiments ( ** p

Techniques Used: Activity Assay, Transfection, Luciferase

Effects of CoPP and curcumin on NQO1 expression. (A, B) HepG2 cells were treated with 10 µM CoPP and 10 µM curcumin for the indicated time period. Total RNA were extracted and expression of NQO1 and GAPDH mRNA was detected by semiquantitative RT-PCR.
Figure Legend Snippet: Effects of CoPP and curcumin on NQO1 expression. (A, B) HepG2 cells were treated with 10 µM CoPP and 10 µM curcumin for the indicated time period. Total RNA were extracted and expression of NQO1 and GAPDH mRNA was detected by semiquantitative RT-PCR.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

Dose-dependent NQO1 mRNA induction by exogenous CO. (A, B) HepG2 cells were treated with various concentrations of CORM-2 as exogenous CO donor or Rucl2 as control for 6 h and the cells were harvested for semi-quantitative RT-PCR. The mRNA expression of NQO1 was determined by semi-quantitative RT-PCR. GAPDH was used as internal controls.
Figure Legend Snippet: Dose-dependent NQO1 mRNA induction by exogenous CO. (A, B) HepG2 cells were treated with various concentrations of CORM-2 as exogenous CO donor or Rucl2 as control for 6 h and the cells were harvested for semi-quantitative RT-PCR. The mRNA expression of NQO1 was determined by semi-quantitative RT-PCR. GAPDH was used as internal controls.

Techniques Used: Quantitative RT-PCR, Expressing

5) Product Images from "Dorsal-zone-specific reduction of sensory neuron density in the olfactory epithelium following long-term exercise or caloric restriction"

Article Title: Dorsal-zone-specific reduction of sensory neuron density in the olfactory epithelium following long-term exercise or caloric restriction

Journal: Scientific Reports

doi: 10.1038/s41598-018-35607-w

Ex- and CR-induced reduction in OSN numbers occur selectively in the NQO1-positive dorsal zone of the olfactory epithelium. (a–c) Photomicrographs of representative coronal sections in control, Ex and CR mice. The rectangular portions of the OE in the left-hand photographs are enlarged in the right-hand images (red, anti-NQO1; green, anti-OMP; blue, DAPI). DM1, upper nasal septum in the dorsomedial area; DM2, upper concha bullosa in the dorsomedial area. Closed triangle, upper border of the NQO1-positive area; open triangle, lower border of the NQO1-positive area. Scale bars: 300 µm at low magnification, 50 µm at higher magnification. (d) The number of NQO1-positive cells in the dorsal zone in control, Ex and CR mice. The number of NQO1-positive cells was significantly lower in Ex and CR mice than in control mice (***p
Figure Legend Snippet: Ex- and CR-induced reduction in OSN numbers occur selectively in the NQO1-positive dorsal zone of the olfactory epithelium. (a–c) Photomicrographs of representative coronal sections in control, Ex and CR mice. The rectangular portions of the OE in the left-hand photographs are enlarged in the right-hand images (red, anti-NQO1; green, anti-OMP; blue, DAPI). DM1, upper nasal septum in the dorsomedial area; DM2, upper concha bullosa in the dorsomedial area. Closed triangle, upper border of the NQO1-positive area; open triangle, lower border of the NQO1-positive area. Scale bars: 300 µm at low magnification, 50 µm at higher magnification. (d) The number of NQO1-positive cells in the dorsal zone in control, Ex and CR mice. The number of NQO1-positive cells was significantly lower in Ex and CR mice than in control mice (***p

Techniques Used: Mouse Assay

Schematic diagram illustrating two parallel olfactory pathways. (a) NQO1-positive OSNs in the dorsal zone of the OE project to the dorsal domain in the main OB. Aversive odor signals are transmitted from the dorsal domain in the main OB (spoiled-food-odor-responsive glomeruli, SF; predator-odor-responsive glomeruli, PO) to the cortical amygdala (CoA), mediating the aversive behavior to spoiled food odors and the fear response to predator odors. NQO1-negative OSNs in the ventral zone project to the ventral domain in the main OB. Attractive social odor signals are transmitted from the posteroventral (PV) part of the main OB to the anterior amygdala (aMeA), mediating attractive behavior to social odor cues. OE, olfactory epithelium; OB, olfactory bulb; OSNs, olfactory sensory neurons. (b) Long-term Ex and CR reduce the number of functional OSNs in the dorsal zone that give rise to the dorsal olfactory pathway. By contrast, long-term Ex and CR have little effect on OSNs in the ventral zone that give rise to the ventral pathway.
Figure Legend Snippet: Schematic diagram illustrating two parallel olfactory pathways. (a) NQO1-positive OSNs in the dorsal zone of the OE project to the dorsal domain in the main OB. Aversive odor signals are transmitted from the dorsal domain in the main OB (spoiled-food-odor-responsive glomeruli, SF; predator-odor-responsive glomeruli, PO) to the cortical amygdala (CoA), mediating the aversive behavior to spoiled food odors and the fear response to predator odors. NQO1-negative OSNs in the ventral zone project to the ventral domain in the main OB. Attractive social odor signals are transmitted from the posteroventral (PV) part of the main OB to the anterior amygdala (aMeA), mediating attractive behavior to social odor cues. OE, olfactory epithelium; OB, olfactory bulb; OSNs, olfactory sensory neurons. (b) Long-term Ex and CR reduce the number of functional OSNs in the dorsal zone that give rise to the dorsal olfactory pathway. By contrast, long-term Ex and CR have little effect on OSNs in the ventral zone that give rise to the ventral pathway.

Techniques Used: Functional Assay

Ex and CR reduce glomerular size and the neuronal response to odorants selectively in the dorsal domain of the olfactory bulb. (a) Area of analysis in the olfactory bulb (OB). Left, coronal section of the OB stained with anti-NQO1 antibody (red) and DAPI (blue). Right, schematic diagram of the OB showing NQO1-positive and -negative areas. (b) Representative coronal sections stained with anti-OMP (green) in control, Ex and CR mice. Each circled area corresponds to a glomerulus. Scale bar, 50 µm. (c) Summary of the ratio of areas stained and unstained with OMP. The OMP-stained area of NQO1-positive OB was significantly smaller in Ex and CR mice than in control mice (***p
Figure Legend Snippet: Ex and CR reduce glomerular size and the neuronal response to odorants selectively in the dorsal domain of the olfactory bulb. (a) Area of analysis in the olfactory bulb (OB). Left, coronal section of the OB stained with anti-NQO1 antibody (red) and DAPI (blue). Right, schematic diagram of the OB showing NQO1-positive and -negative areas. (b) Representative coronal sections stained with anti-OMP (green) in control, Ex and CR mice. Each circled area corresponds to a glomerulus. Scale bar, 50 µm. (c) Summary of the ratio of areas stained and unstained with OMP. The OMP-stained area of NQO1-positive OB was significantly smaller in Ex and CR mice than in control mice (***p

Techniques Used: Staining, Mouse Assay

6) Product Images from "Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells"

Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2018.9288

Effect of KEAP1 knockdown on the expression of NRF2 and downstream targets. (A) mRNA expression levels of NRF2, NQO1 and HO1 in Hep2, scHep2 and shKEAP1 Hep2 cells. Expression levels of NRF2, NQO1 and HO1 were increased following the knockdown of KEAP1 in Hep2 cells. (B) Representative NRF2 immunofluorescence staining images indicate that NRF2 translocated into the nuclei from the cytoplasm following knockdown of KEAP1 in Hep2 cells (magnification, ×40). (C) Western blotting demonstrated that nuclear NRF2 protein expression levels were elevated, while cytoplasmic NRF2 protein expression levels were reduced, following the knockdown of KEAP1 in Hep2 cells. (D) Western blotting demonstrated that total NQO1 and HO1 protein expression levels were increased within shKEAP1 Hep2 cells, compared with the scHep2 group. *P
Figure Legend Snippet: Effect of KEAP1 knockdown on the expression of NRF2 and downstream targets. (A) mRNA expression levels of NRF2, NQO1 and HO1 in Hep2, scHep2 and shKEAP1 Hep2 cells. Expression levels of NRF2, NQO1 and HO1 were increased following the knockdown of KEAP1 in Hep2 cells. (B) Representative NRF2 immunofluorescence staining images indicate that NRF2 translocated into the nuclei from the cytoplasm following knockdown of KEAP1 in Hep2 cells (magnification, ×40). (C) Western blotting demonstrated that nuclear NRF2 protein expression levels were elevated, while cytoplasmic NRF2 protein expression levels were reduced, following the knockdown of KEAP1 in Hep2 cells. (D) Western blotting demonstrated that total NQO1 and HO1 protein expression levels were increased within shKEAP1 Hep2 cells, compared with the scHep2 group. *P

Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot

Effect of KEAP1 knockdown on H 2 O 2 -induced alterations in the expression of NQO1 and HO1. mRNA levels of (A) NQO1 and (B) HO1 within shKEAP1 Hep2 cells were increased in a dose- and time-dependent manner following exposure to H 2 O 2 . KEAP1 knockdown upregulated the expression levels of NQO1 and HO1 compared with in scHep2 cells. *P
Figure Legend Snippet: Effect of KEAP1 knockdown on H 2 O 2 -induced alterations in the expression of NQO1 and HO1. mRNA levels of (A) NQO1 and (B) HO1 within shKEAP1 Hep2 cells were increased in a dose- and time-dependent manner following exposure to H 2 O 2 . KEAP1 knockdown upregulated the expression levels of NQO1 and HO1 compared with in scHep2 cells. *P

Techniques Used: Expressing

7) Product Images from "NRF2-regulated cell cycle arrest at early stage of oxidative stress response mechanism"

Article Title: NRF2-regulated cell cycle arrest at early stage of oxidative stress response mechanism

Journal: PLoS ONE

doi: 10.1371/journal.pone.0207949

Silencing of NRF2 down-regulates Cyclin D1 inhibitors at early stage of oxidative stress. Time dependency of oxidative stress treatment was followed with/without a background of NRF2 silencing. HEK293T cells were cultured with TBHP for 0.5-1-2-3 hours, while NRF2 gene expression was depleted by NRF2 siRNA. (A) Testing the effectiveness of RNA silencing. NRF2 mRNA level was followed by real-time PCR in cell culture containing NRF2 siRNA. GAPDH was used as a housekeeping gene. The intensity of NRF2 is normalised for GAPDH. (B) During oxidative stress the markers of NRF2 (NQO1), PERK (PERK-T, eiF2α -P), Cyclin D1 and its inhibitors (p15, p16, p21, p27) were followed by immunoblotting. GAPDH was used as a loading control. (C) Densitometry data represent the intensity of NQO1, PERK-T, Cyclin D1, p15, p16, p21 and p27 normalised for GAPDH and eiF2α-P normalized for total level of eiF2α. For each of the experiments, three independent measurements were carried out. Error bars represent standard deviation; asterisks indicate statistically significant difference from the control: * p
Figure Legend Snippet: Silencing of NRF2 down-regulates Cyclin D1 inhibitors at early stage of oxidative stress. Time dependency of oxidative stress treatment was followed with/without a background of NRF2 silencing. HEK293T cells were cultured with TBHP for 0.5-1-2-3 hours, while NRF2 gene expression was depleted by NRF2 siRNA. (A) Testing the effectiveness of RNA silencing. NRF2 mRNA level was followed by real-time PCR in cell culture containing NRF2 siRNA. GAPDH was used as a housekeeping gene. The intensity of NRF2 is normalised for GAPDH. (B) During oxidative stress the markers of NRF2 (NQO1), PERK (PERK-T, eiF2α -P), Cyclin D1 and its inhibitors (p15, p16, p21, p27) were followed by immunoblotting. GAPDH was used as a loading control. (C) Densitometry data represent the intensity of NQO1, PERK-T, Cyclin D1, p15, p16, p21 and p27 normalised for GAPDH and eiF2α-P normalized for total level of eiF2α. For each of the experiments, three independent measurements were carried out. Error bars represent standard deviation; asterisks indicate statistically significant difference from the control: * p

Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

Short treatment with high level of oxidative stress induces Cyclin D1 inhibitors. (A) HEK293T cells were treated with 100 or 300 μM TBHP for 1.5 hour and the markers of NRF2 (NQO1), PERK (PERK-T, eiF2α -P), Cyclin D1 and its inhibitors (p15, p16, p21, p27) were followed by immunoblotting. GAPDH was used as a loading control. (B) Densitometry data represent the intensity of NQO1, PERK-T, Cyclin D1, p15, p16, p21 and p27 normalised for GAPDH and eiF2α-P normalized for total level of eiF2α. For each of the experiments, three independent measurements were carried out. Error bars represent standard deviation; asterisks indicate statistically significant difference from the control: * p
Figure Legend Snippet: Short treatment with high level of oxidative stress induces Cyclin D1 inhibitors. (A) HEK293T cells were treated with 100 or 300 μM TBHP for 1.5 hour and the markers of NRF2 (NQO1), PERK (PERK-T, eiF2α -P), Cyclin D1 and its inhibitors (p15, p16, p21, p27) were followed by immunoblotting. GAPDH was used as a loading control. (B) Densitometry data represent the intensity of NQO1, PERK-T, Cyclin D1, p15, p16, p21 and p27 normalised for GAPDH and eiF2α-P normalized for total level of eiF2α. For each of the experiments, three independent measurements were carried out. Error bars represent standard deviation; asterisks indicate statistically significant difference from the control: * p

Techniques Used: Standard Deviation

Silencing of PERK down-regulates Cyclin D1 inhibitors at early stage of oxidative stress. Time dependency of oxidative stress treatment was followed with/without a background of PERK silencing. HEK293T cells were cultured with TBHP for 0.5-1-2-3 hours, while PERK gene expression was depleted by PERK siRNA. (A) Testing the effectiveness of RNA silencing. PERK mRNA level was followed by real-time PCR in cell culture containing PERK siRNA. GAPDH was used as a housekeeping gene. The intensity of PERK is normalised for GAPDH. (B) During oxidative stress the markers of NRF2 (NQO1), PERK (PERK-T, eiF2α -P), Cyclin D1 and its inhibitors (p15, p16, p21, p27) were followed by immunoblotting. GAPDH was used as a loading control. (C) Densitometry data represent the intensity of NQO1, PERK-T, Cyclin D1, p15, p16, p21 and p27 normalised for GAPDH and eiF2α-P normalized for total level of eiF2α. For each of the experiments, three independent measurements were carried out. Error bars represent standard deviation; asterisks indicate statistically significant difference from the control: * p
Figure Legend Snippet: Silencing of PERK down-regulates Cyclin D1 inhibitors at early stage of oxidative stress. Time dependency of oxidative stress treatment was followed with/without a background of PERK silencing. HEK293T cells were cultured with TBHP for 0.5-1-2-3 hours, while PERK gene expression was depleted by PERK siRNA. (A) Testing the effectiveness of RNA silencing. PERK mRNA level was followed by real-time PCR in cell culture containing PERK siRNA. GAPDH was used as a housekeeping gene. The intensity of PERK is normalised for GAPDH. (B) During oxidative stress the markers of NRF2 (NQO1), PERK (PERK-T, eiF2α -P), Cyclin D1 and its inhibitors (p15, p16, p21, p27) were followed by immunoblotting. GAPDH was used as a loading control. (C) Densitometry data represent the intensity of NQO1, PERK-T, Cyclin D1, p15, p16, p21 and p27 normalised for GAPDH and eiF2α-P normalized for total level of eiF2α. For each of the experiments, three independent measurements were carried out. Error bars represent standard deviation; asterisks indicate statistically significant difference from the control: * p

Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

Cyclin D1 degradation is not observed at early stage of low level of oxidative stress. Concentration dependency of oxidative stress treatment was followed with/without a background of PERK or NRF2 silencing. HEK293T cells were cultured with 100 or 300 μM TBHP for 1.5 hours, while NRF2 or PERK gene expression was depleted by NRF2 or PERK siRNA. ( A) The relative amount of viable HEK293T cells. (B) The efficiency of NRF2 (upper panel) and PERK (lower panel) silencing was checked on mRNA level. The mRNA level was followed by real-time PCR. GAPDH was used as a housekeeping gene. The intensity of NRF2 is normalised for GAPDH. (C) During oxidative stress the markers of NRF2 (NQO1), PERK (PERK-T, eiF2α -P) and Cyclin D1 were followed by immunoblotting. GAPDH was used as a loading control. (D) Densitometry data represent the intensity of NQO1, PERK-T and Cyclin D1 normalised for GAPDH and eiF2α-P normalized for total level of eiF2α. For each of the experiments, three independent measurements were carried out. Error bars represent standard deviation; asterisks indicate statistically significant difference from the control: * p
Figure Legend Snippet: Cyclin D1 degradation is not observed at early stage of low level of oxidative stress. Concentration dependency of oxidative stress treatment was followed with/without a background of PERK or NRF2 silencing. HEK293T cells were cultured with 100 or 300 μM TBHP for 1.5 hours, while NRF2 or PERK gene expression was depleted by NRF2 or PERK siRNA. ( A) The relative amount of viable HEK293T cells. (B) The efficiency of NRF2 (upper panel) and PERK (lower panel) silencing was checked on mRNA level. The mRNA level was followed by real-time PCR. GAPDH was used as a housekeeping gene. The intensity of NRF2 is normalised for GAPDH. (C) During oxidative stress the markers of NRF2 (NQO1), PERK (PERK-T, eiF2α -P) and Cyclin D1 were followed by immunoblotting. GAPDH was used as a loading control. (D) Densitometry data represent the intensity of NQO1, PERK-T and Cyclin D1 normalised for GAPDH and eiF2α-P normalized for total level of eiF2α. For each of the experiments, three independent measurements were carried out. Error bars represent standard deviation; asterisks indicate statistically significant difference from the control: * p

Techniques Used: Concentration Assay, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

8) Product Images from "Dorsal-zone-specific reduction of sensory neuron density in the olfactory epithelium following long-term exercise or caloric restriction"

Article Title: Dorsal-zone-specific reduction of sensory neuron density in the olfactory epithelium following long-term exercise or caloric restriction

Journal: Scientific Reports

doi: 10.1038/s41598-018-35607-w

Ex- and CR-induced reduction in OSN numbers occur selectively in the NQO1-positive dorsal zone of the olfactory epithelium. (a–c) Photomicrographs of representative coronal sections in control, Ex and CR mice. The rectangular portions of the OE in the left-hand photographs are enlarged in the right-hand images (red, anti-NQO1; green, anti-OMP; blue, DAPI). DM1, upper nasal septum in the dorsomedial area; DM2, upper concha bullosa in the dorsomedial area. Closed triangle, upper border of the NQO1-positive area; open triangle, lower border of the NQO1-positive area. Scale bars: 300 µm at low magnification, 50 µm at higher magnification. (d) The number of NQO1-positive cells in the dorsal zone in control, Ex and CR mice. The number of NQO1-positive cells was significantly lower in Ex and CR mice than in control mice (***p
Figure Legend Snippet: Ex- and CR-induced reduction in OSN numbers occur selectively in the NQO1-positive dorsal zone of the olfactory epithelium. (a–c) Photomicrographs of representative coronal sections in control, Ex and CR mice. The rectangular portions of the OE in the left-hand photographs are enlarged in the right-hand images (red, anti-NQO1; green, anti-OMP; blue, DAPI). DM1, upper nasal septum in the dorsomedial area; DM2, upper concha bullosa in the dorsomedial area. Closed triangle, upper border of the NQO1-positive area; open triangle, lower border of the NQO1-positive area. Scale bars: 300 µm at low magnification, 50 µm at higher magnification. (d) The number of NQO1-positive cells in the dorsal zone in control, Ex and CR mice. The number of NQO1-positive cells was significantly lower in Ex and CR mice than in control mice (***p

Techniques Used: Mouse Assay

Ex and CR reduce glomerular size and the neuronal response to odorants selectively in the dorsal domain of the olfactory bulb. (a) Area of analysis in the olfactory bulb (OB). Left, coronal section of the OB stained with anti-NQO1 antibody (red) and DAPI (blue). Right, schematic diagram of the OB showing NQO1-positive and -negative areas. (b) Representative coronal sections stained with anti-OMP (green) in control, Ex and CR mice. Each circled area corresponds to a glomerulus. Scale bar, 50 µm. (c) Summary of the ratio of areas stained and unstained with OMP. The OMP-stained area of NQO1-positive OB was significantly smaller in Ex and CR mice than in control mice (***p
Figure Legend Snippet: Ex and CR reduce glomerular size and the neuronal response to odorants selectively in the dorsal domain of the olfactory bulb. (a) Area of analysis in the olfactory bulb (OB). Left, coronal section of the OB stained with anti-NQO1 antibody (red) and DAPI (blue). Right, schematic diagram of the OB showing NQO1-positive and -negative areas. (b) Representative coronal sections stained with anti-OMP (green) in control, Ex and CR mice. Each circled area corresponds to a glomerulus. Scale bar, 50 µm. (c) Summary of the ratio of areas stained and unstained with OMP. The OMP-stained area of NQO1-positive OB was significantly smaller in Ex and CR mice than in control mice (***p

Techniques Used: Staining, Mouse Assay

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Centrifugation:

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Blocking Assay:

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Article Snippet: .. To block the membranes, 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.4–7.5) was applied at room temperature for 1 h. Membranes were subsequently incubated overnight with anti-NRF2 (cat. no. ab31163; Abcam), anti-KEAP1 (cat. no. ab139729; Abcam), anti-NQO1 (cat. no. A180; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-HO1 (cat. no. ab68477; Abcam), and anti-β-actin (cat. no. GB13001-3; Servicebio, Inc.) or lamin B primary antibodies (GB11408; Servicebio, Inc.) in blocking buffer at 4°C at 1:1,000 dilution. .. Subsequently, membranes were washed three times in TBST followed by incubation for 1 h with HRP-labeled goat anti-rabbit/mouse IgG (H+L) (GB23303/GB23301; Servicebio, Inc.) at a 1:2,500 dilution and washed in TBST again.

Enzyme-linked Immunosorbent Assay:

Article Title: NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice
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Incubation:

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Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells
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Article Title: Dorsal-zone-specific reduction of sensory neuron density in the olfactory epithelium following long-term exercise or caloric restriction
Article Snippet: Immunohistochemistry was performed with the following antibodies: anti-olfactory marker protein (OMP, goat polyclonal, 1:2000 dilution; Wako Chemicals), anti-NQO1 (rabbit polyclonal, 1:300; Cell Signaling Technology), anti-activated caspase-3 (rabbit polyclonal, 1:300; Cell Signaling Technology), anti-Ki67 (mouse monoclonal, 1:300; BD Biosciences), anti-c-fos (rabbit polyclonal, 1:50; Santa Cruz Biotechnology), anti-MnSOD (rabbit monoclonal, 1:100; Epitomics Inc.), and anti-8-hydroxy-2′-deoxyguanosine (8-OHdG, goat polyclonal antibody, 1:100; Alpha Diagnostic International Inc.). .. Immunoreactivity was detected using the following antibodies in the Histofine Simple StainMAX-PO secondary antibody system (Nichirei) according to the manufacturers’ instructions, donkey anti-goat Alexa Fluor 488 (1:100; Invitrogen), and donkey anti-rabbit Alexa Fluor 594 (1:100; Invitrogen), incubated for 1 h at room temperature.

BIA-KA:

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Article Snippet: Protein was quantified via the bicinchoninic acid assay (BCA protein assay kit; Pierce). .. Aliquots containing 15 μg protein were mixed with 2× reducing sample buffer (containing 4% SDS, 20% glycerol, and 10% β-mercaptoethanol), and separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for specific proteins with the following primary antibodies: TrxR1: (1:1000, Abcam), Trx1 (1:2000, Cell Signaling), phospho SAPK/JNK1/2 (1:1000, Cell Signaling), SAPK/JNK1/2 (1:1000, Cell Signaling), phospho ERK1/2 (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho P-38 (1:1000, Cell Signaling), P-38 (1:1000, Cell Signaling), HO-1 (1:250, Biovision), NQO1 (1:1000, Cell Signaling), β-actin (1:5000, Sigma).

Modification:

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Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and 0.05% trypsin and other cell culture reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. Antibodies listed below were used: anti-Nrf2 (Cell Signaling, Beverly, MA, USA, #12721), anti-NQO1 (Cell Signaling, #3187), anti-Bax (Cell Signaling, #2772), anti-Bcl2 (Cell Signaling, #2876), anti-cleaved caspase 3 (17 kDa) (Cell Signaling, #9664), anti-HO-1 (Cell Signaling, #5061), anti-Grx1 (Abcam, Cambridge, MA, USA, ab45953), anti-Trx1 (Abcam, ab86255), anti-PSSG (Virogen, Watertown, MA, USA, 101-A-100), anti-SOD2 (signal, HPA001814), anti-catalase (Abcam, ab16731), anti-GAPDH (Santa Cruz, Santa Cruz, CA, USA, sc-32233), anti-B23 antibodies, and horseradish peroxidase-conjugated secondary antibodies (sc2061, sc2060, sc2030; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Western Blot:

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Article Title: NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice
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Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells
Article Snippet: Paragraph title: Western blot analysis ... To block the membranes, 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.4–7.5) was applied at room temperature for 1 h. Membranes were subsequently incubated overnight with anti-NRF2 (cat. no. ab31163; Abcam), anti-KEAP1 (cat. no. ab139729; Abcam), anti-NQO1 (cat. no. A180; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-HO1 (cat. no. ab68477; Abcam), and anti-β-actin (cat. no. GB13001-3; Servicebio, Inc.) or lamin B primary antibodies (GB11408; Servicebio, Inc.) in blocking buffer at 4°C at 1:1,000 dilution.

Immunohistochemistry:

Article Title: Dorsal-zone-specific reduction of sensory neuron density in the olfactory epithelium following long-term exercise or caloric restriction
Article Snippet: .. Immunohistochemistry was performed with the following antibodies: anti-olfactory marker protein (OMP, goat polyclonal, 1:2000 dilution; Wako Chemicals), anti-NQO1 (rabbit polyclonal, 1:300; Cell Signaling Technology), anti-activated caspase-3 (rabbit polyclonal, 1:300; Cell Signaling Technology), anti-Ki67 (mouse monoclonal, 1:300; BD Biosciences), anti-c-fos (rabbit polyclonal, 1:50; Santa Cruz Biotechnology), anti-MnSOD (rabbit monoclonal, 1:100; Epitomics Inc.), and anti-8-hydroxy-2′-deoxyguanosine (8-OHdG, goat polyclonal antibody, 1:100; Alpha Diagnostic International Inc.). .. Immunoreactivity was detected using the following antibodies in the Histofine Simple StainMAX-PO secondary antibody system (Nichirei) according to the manufacturers’ instructions, donkey anti-goat Alexa Fluor 488 (1:100; Invitrogen), and donkey anti-rabbit Alexa Fluor 594 (1:100; Invitrogen), incubated for 1 h at room temperature.

Activation Assay:

Article Title: NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice
Article Snippet: .. Analysis of Nrf2 Activation Western blot analysis of nuclear protein fractions was performed by Odyssey system (LI-COR Bioscience, Nebraska USA), using antibodies specific for Nrf2, TBP, beta-actin (Santa Cruz Technology), NQO1 (Cell Signaling Technology). .. In whole lung nuclear protein extracts, Nrf2 was measured by the TransAM Nrf2 ELISA (Active Motif, Carlsbad, CA) using the manufacturers instructions.

Cell Culture:

Article Title: The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2) activation
Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and 0.05% trypsin and other cell culture reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. Antibodies listed below were used: anti-Nrf2 (Cell Signaling, Beverly, MA, USA, #12721), anti-NQO1 (Cell Signaling, #3187), anti-Bax (Cell Signaling, #2772), anti-Bcl2 (Cell Signaling, #2876), anti-cleaved caspase 3 (17 kDa) (Cell Signaling, #9664), anti-HO-1 (Cell Signaling, #5061), anti-Grx1 (Abcam, Cambridge, MA, USA, ab45953), anti-Trx1 (Abcam, ab86255), anti-PSSG (Virogen, Watertown, MA, USA, 101-A-100), anti-SOD2 (signal, HPA001814), anti-catalase (Abcam, ab16731), anti-GAPDH (Santa Cruz, Santa Cruz, CA, USA, sc-32233), anti-B23 antibodies, and horseradish peroxidase-conjugated secondary antibodies (sc2061, sc2060, sc2030; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

other:

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Imaging:

Article Title: Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2
Article Snippet: The primary antibodies used were: anti-tubulin (catalog no.: N-356, Amersham, GE Healthcare, Pittsburgh, PA), anti-Nrf2 (catalog no.: sc-13032, Santa Cruz Biotechnology, Inc., Dallas, TX) anti-SAMHD1 (catalog no.: GTX83687; GeneTex, Irvine, CA), anti-NQO1 (catalog no.: 3187S; Cell Signaling Technologies, Danvers, MA), anti-GCLM (catalog no.: GTX114075; GeneTex, Irvine, CA), anti-Mx2 (catalog no.: D0214; Santa Cruz Biotechnology, Inc., Dallas, TX) and anti-HIV-1 p24 (catalog no.: 183–H12-5C, obtained from Bruce Chesebro and Hardy Chen through the NIH AIDS Research and Reference Reagent Program). .. Chemiluminescent blot imaging was done with an Alpha Innotech FluorChem HD2 Imaging system.

Sonication:

Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1
Article Snippet: Following 15 min incubation on ice, cells were sonicated for 20 pulses on ice using a sonic dismembrator (Fisher Scientific) and centrifuged at 14,000 rpm, 4 °C for 5 min to remove cell debris. .. Aliquots containing 15 μg protein were mixed with 2× reducing sample buffer (containing 4% SDS, 20% glycerol, and 10% β-mercaptoethanol), and separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for specific proteins with the following primary antibodies: TrxR1: (1:1000, Abcam), Trx1 (1:2000, Cell Signaling), phospho SAPK/JNK1/2 (1:1000, Cell Signaling), SAPK/JNK1/2 (1:1000, Cell Signaling), phospho ERK1/2 (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho P-38 (1:1000, Cell Signaling), P-38 (1:1000, Cell Signaling), HO-1 (1:250, Biovision), NQO1 (1:1000, Cell Signaling), β-actin (1:5000, Sigma).

Bicinchoninic Acid Protein Assay:

Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells
Article Snippet: Protein extracts were obtained via centrifugation at 4°C and 10,000 × g for 10 min; proteins were quantified using a Bicinchoninic Acid Protein assay kit (Beyotime Institute of Biotechnology). .. To block the membranes, 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.4–7.5) was applied at room temperature for 1 h. Membranes were subsequently incubated overnight with anti-NRF2 (cat. no. ab31163; Abcam), anti-KEAP1 (cat. no. ab139729; Abcam), anti-NQO1 (cat. no. A180; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-HO1 (cat. no. ab68477; Abcam), and anti-β-actin (cat. no. GB13001-3; Servicebio, Inc.) or lamin B primary antibodies (GB11408; Servicebio, Inc.) in blocking buffer at 4°C at 1:1,000 dilution.

Mouse Assay:

Article Title: Dorsal-zone-specific reduction of sensory neuron density in the olfactory epithelium following long-term exercise or caloric restriction
Article Snippet: Immunohistochemistry Mice were perfused with 4% paraformaldehyde in 0.1% phosphate buffer and post-fixed for 24 h in the same fixative. .. Immunohistochemistry was performed with the following antibodies: anti-olfactory marker protein (OMP, goat polyclonal, 1:2000 dilution; Wako Chemicals), anti-NQO1 (rabbit polyclonal, 1:300; Cell Signaling Technology), anti-activated caspase-3 (rabbit polyclonal, 1:300; Cell Signaling Technology), anti-Ki67 (mouse monoclonal, 1:300; BD Biosciences), anti-c-fos (rabbit polyclonal, 1:50; Santa Cruz Biotechnology), anti-MnSOD (rabbit monoclonal, 1:100; Epitomics Inc.), and anti-8-hydroxy-2′-deoxyguanosine (8-OHdG, goat polyclonal antibody, 1:100; Alpha Diagnostic International Inc.).

Polyacrylamide Gel Electrophoresis:

Article Title: NRF2-regulated cell cycle arrest at early stage of oxidative stress response mechanism
Article Snippet: Paragraph title: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis ... SDS-PAGE and Western Blot analysis was done according to our previous study [ ]The following antibodies were applied: anti-NQO1 (Cell Signaling, A180), anti-CyclinD1 (Santa Cruz, A-12), anti-p21 (Santa Cruz, C-19), anti-p27 (Santa Cruz, C-19), anti-p15-INK4b (proteintech, 12877-1-AP), anti-p16-INK4A (proteintech, 10883-1-AP), anti-PERK (Cell Signaling, 3192S), anti-P-eIF2α (Cell Signaling, 9722S9), anti-P-eIF2α (Ser51) (Cell Signaling, 9721L), and anti-GAPDH (Santa Cruz, 6C5) and HRP conjugated secondary antibodies (SantaCruz, sc-2020 and Cell Signaling, 7074S, 7076S).

SDS Page:

Article Title: CO/HO-1 Induces NQO-1 Expression via Nrf2 Activation
Article Snippet: Equal amounts of cell lysates were separated by SDS-PAGE and transferred onto Immobilon-P membranes (Millipore, Bedford, MA). .. The transferred membranes were incubated with anti-NQO1 (Cell Signaling, DE), polyclonal anti-HO-1 (Enzo Life Sciences, NY), anti-actin (Santa Cruz Biotechnology, CA) antibody.

Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1
Article Snippet: .. Aliquots containing 15 μg protein were mixed with 2× reducing sample buffer (containing 4% SDS, 20% glycerol, and 10% β-mercaptoethanol), and separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for specific proteins with the following primary antibodies: TrxR1: (1:1000, Abcam), Trx1 (1:2000, Cell Signaling), phospho SAPK/JNK1/2 (1:1000, Cell Signaling), SAPK/JNK1/2 (1:1000, Cell Signaling), phospho ERK1/2 (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho P-38 (1:1000, Cell Signaling), P-38 (1:1000, Cell Signaling), HO-1 (1:250, Biovision), NQO1 (1:1000, Cell Signaling), β-actin (1:5000, Sigma). .. Primary antibodies were detected using HRP-conjugated anti-rabbit or anti-mouse IgG and visualized by SuperSignal West chemiluminescent substrate (Pierce).

Article Title: NRF2-regulated cell cycle arrest at early stage of oxidative stress response mechanism
Article Snippet: .. SDS-PAGE and Western Blot analysis was done according to our previous study [ ]The following antibodies were applied: anti-NQO1 (Cell Signaling, A180), anti-CyclinD1 (Santa Cruz, A-12), anti-p21 (Santa Cruz, C-19), anti-p27 (Santa Cruz, C-19), anti-p15-INK4b (proteintech, 12877-1-AP), anti-p16-INK4A (proteintech, 10883-1-AP), anti-PERK (Cell Signaling, 3192S), anti-P-eIF2α (Cell Signaling, 9722S9), anti-P-eIF2α (Ser51) (Cell Signaling, 9721L), and anti-GAPDH (Santa Cruz, 6C5) and HRP conjugated secondary antibodies (SantaCruz, sc-2020 and Cell Signaling, 7074S, 7076S). .. Statistics For densitometry analysis, Western blot data were acquired using ImageJ software, while the statistical analysis was performed as described previously [ ].

Article Title: Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2
Article Snippet: Immunoblotting Proteins from cells lysed with Laemmli buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 0.1% bromophenol blue) were resolved by SDS-PAGE, transferred onto PVDF membrane (Millipore, Billerica, MA) and probed with the indicated antibodies. .. The primary antibodies used were: anti-tubulin (catalog no.: N-356, Amersham, GE Healthcare, Pittsburgh, PA), anti-Nrf2 (catalog no.: sc-13032, Santa Cruz Biotechnology, Inc., Dallas, TX) anti-SAMHD1 (catalog no.: GTX83687; GeneTex, Irvine, CA), anti-NQO1 (catalog no.: 3187S; Cell Signaling Technologies, Danvers, MA), anti-GCLM (catalog no.: GTX114075; GeneTex, Irvine, CA), anti-Mx2 (catalog no.: D0214; Santa Cruz Biotechnology, Inc., Dallas, TX) and anti-HIV-1 p24 (catalog no.: 183–H12-5C, obtained from Bruce Chesebro and Hardy Chen through the NIH AIDS Research and Reference Reagent Program).

Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells
Article Snippet: A total of 40 µg total protein was electrophoretically separated via 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. .. To block the membranes, 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.4–7.5) was applied at room temperature for 1 h. Membranes were subsequently incubated overnight with anti-NRF2 (cat. no. ab31163; Abcam), anti-KEAP1 (cat. no. ab139729; Abcam), anti-NQO1 (cat. no. A180; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-HO1 (cat. no. ab68477; Abcam), and anti-β-actin (cat. no. GB13001-3; Servicebio, Inc.) or lamin B primary antibodies (GB11408; Servicebio, Inc.) in blocking buffer at 4°C at 1:1,000 dilution.

Software:

Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1
Article Snippet: Aliquots containing 15 μg protein were mixed with 2× reducing sample buffer (containing 4% SDS, 20% glycerol, and 10% β-mercaptoethanol), and separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for specific proteins with the following primary antibodies: TrxR1: (1:1000, Abcam), Trx1 (1:2000, Cell Signaling), phospho SAPK/JNK1/2 (1:1000, Cell Signaling), SAPK/JNK1/2 (1:1000, Cell Signaling), phospho ERK1/2 (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho P-38 (1:1000, Cell Signaling), P-38 (1:1000, Cell Signaling), HO-1 (1:250, Biovision), NQO1 (1:1000, Cell Signaling), β-actin (1:5000, Sigma). .. Band densities were evaluated using ImageJ software, and normalized to total levels of respective proteins or to β-actin.

Article Title: The Unfolded Protein Response Regulates Uterine Myocyte Antioxidant Responsiveness During Pregnancy 1
Article Snippet: The concentrations of primary antibodies and their sources are as follows: 78 kDa glucose regulated protein (GRP78; 1:1000, #CST-9271) growth arrest and DNA damage inducible protein 152 (GADD153; 1:500, #CST-5554), caspase 3 (CASP3; 1:200, #CST-9662), poly ADP ribose polymerase (PARP; 1:200, #CST-5625), EIF2AK3 (1:250, #CST-3192), pEIF2AK3 (1:250, #CST-3179), Cl.CASP3 (1:500, #CST-9664), NFE2L2 (1:250, #CST-12721), NQO1 (1:5000, #CST-3187), NF kappa B p65 subunit (p65; 1:500 #CST-3034), and PDIA2 (1:10000, #3501), were obtained from Cell Signaling Technology. .. The immunoreactive bands were quantified using ImageJ software and normalized to NCOA3 and PDIA2 for nuclear and cytosolic fractions, respectively.

Article Title: Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2
Article Snippet: The primary antibodies used were: anti-tubulin (catalog no.: N-356, Amersham, GE Healthcare, Pittsburgh, PA), anti-Nrf2 (catalog no.: sc-13032, Santa Cruz Biotechnology, Inc., Dallas, TX) anti-SAMHD1 (catalog no.: GTX83687; GeneTex, Irvine, CA), anti-NQO1 (catalog no.: 3187S; Cell Signaling Technologies, Danvers, MA), anti-GCLM (catalog no.: GTX114075; GeneTex, Irvine, CA), anti-Mx2 (catalog no.: D0214; Santa Cruz Biotechnology, Inc., Dallas, TX) and anti-HIV-1 p24 (catalog no.: 183–H12-5C, obtained from Bruce Chesebro and Hardy Chen through the NIH AIDS Research and Reference Reagent Program). .. Densitometric analysis was performed using the ImageJ image analysis software from the NIH.

Electrophoresis:

Article Title: The Unfolded Protein Response Regulates Uterine Myocyte Antioxidant Responsiveness During Pregnancy 1
Article Snippet: NuPAGE precast 4%–12% gradient gels (Life Technologies) were used for electrophoresis, and protein was transferred onto Hybond-P polyvinylidene fluoride (PVDF) membranes (Millipore). .. The concentrations of primary antibodies and their sources are as follows: 78 kDa glucose regulated protein (GRP78; 1:1000, #CST-9271) growth arrest and DNA damage inducible protein 152 (GADD153; 1:500, #CST-5554), caspase 3 (CASP3; 1:200, #CST-9662), poly ADP ribose polymerase (PARP; 1:200, #CST-5625), EIF2AK3 (1:250, #CST-3192), pEIF2AK3 (1:250, #CST-3179), Cl.CASP3 (1:500, #CST-9664), NFE2L2 (1:250, #CST-12721), NQO1 (1:5000, #CST-3187), NF kappa B p65 subunit (p65; 1:500 #CST-3034), and PDIA2 (1:10000, #3501), were obtained from Cell Signaling Technology.

Acid Assay:

Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1
Article Snippet: Protein was quantified via the bicinchoninic acid assay (BCA protein assay kit; Pierce). .. Aliquots containing 15 μg protein were mixed with 2× reducing sample buffer (containing 4% SDS, 20% glycerol, and 10% β-mercaptoethanol), and separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for specific proteins with the following primary antibodies: TrxR1: (1:1000, Abcam), Trx1 (1:2000, Cell Signaling), phospho SAPK/JNK1/2 (1:1000, Cell Signaling), SAPK/JNK1/2 (1:1000, Cell Signaling), phospho ERK1/2 (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho P-38 (1:1000, Cell Signaling), P-38 (1:1000, Cell Signaling), HO-1 (1:250, Biovision), NQO1 (1:1000, Cell Signaling), β-actin (1:5000, Sigma).

Concentration Assay:

Article Title: Activation of the KEAP1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells
Article Snippet: Total protein was acquired by lysing cells in a radioimmunoprecicpitation assay buffer containing phenylmethylsulfonyl fluoride with a final concentration of 1 mmol/l (Beijing Cowin Biotech Co., Ltd., Beijing, China). .. To block the membranes, 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.4–7.5) was applied at room temperature for 1 h. Membranes were subsequently incubated overnight with anti-NRF2 (cat. no. ab31163; Abcam), anti-KEAP1 (cat. no. ab139729; Abcam), anti-NQO1 (cat. no. A180; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-HO1 (cat. no. ab68477; Abcam), and anti-β-actin (cat. no. GB13001-3; Servicebio, Inc.) or lamin B primary antibodies (GB11408; Servicebio, Inc.) in blocking buffer at 4°C at 1:1,000 dilution.

Marker:

Article Title: Dorsal-zone-specific reduction of sensory neuron density in the olfactory epithelium following long-term exercise or caloric restriction
Article Snippet: .. Immunohistochemistry was performed with the following antibodies: anti-olfactory marker protein (OMP, goat polyclonal, 1:2000 dilution; Wako Chemicals), anti-NQO1 (rabbit polyclonal, 1:300; Cell Signaling Technology), anti-activated caspase-3 (rabbit polyclonal, 1:300; Cell Signaling Technology), anti-Ki67 (mouse monoclonal, 1:300; BD Biosciences), anti-c-fos (rabbit polyclonal, 1:50; Santa Cruz Biotechnology), anti-MnSOD (rabbit monoclonal, 1:100; Epitomics Inc.), and anti-8-hydroxy-2′-deoxyguanosine (8-OHdG, goat polyclonal antibody, 1:100; Alpha Diagnostic International Inc.). .. Immunoreactivity was detected using the following antibodies in the Histofine Simple StainMAX-PO secondary antibody system (Nichirei) according to the manufacturers’ instructions, donkey anti-goat Alexa Fluor 488 (1:100; Invitrogen), and donkey anti-rabbit Alexa Fluor 594 (1:100; Invitrogen), incubated for 1 h at room temperature.

Lysis:

Article Title: CO/HO-1 Induces NQO-1 Expression via Nrf2 Activation
Article Snippet: Immunoblot For immunoblot, cells were lysed in a lysis buffer containing 25 mM Tris-HCl, pH 7.5, 137 mM NaCl, 2.7 mM KCl, 1% Triton X-100, and Halt™ Protease and Phosphatase inhibitor cocktail (Thermo Scientific, IL), and spun to separate insoluble debris from the clear lysates. .. The transferred membranes were incubated with anti-NQO1 (Cell Signaling, DE), polyclonal anti-HO-1 (Enzo Life Sciences, NY), anti-actin (Santa Cruz Biotechnology, CA) antibody.

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  • 91
    Cell Signaling Technology Inc antibody against nqo1
    Association between <t>NQO1</t> expression and β-lap cytotoxicity in melanoma cell lines. Levels of NQO1 expression determined by (A) western blotting and (B) viability following treatment with β-lap with or without simultaneous treatment with auxin in an AID-NQO1-expressing cell line. NQO1 expression levels in CRL-1585 cells determined by (C) western blotting, and (D) their viability following treatment with β-lap in comparison with cells treated with control siRNA or siRNA against NQO1. The results are presented as mean ± standard deviation from triplicate experiments (*P
    Antibody Against Nqo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibody against nqo1/product/Cell Signaling Technology Inc
    Average 91 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    antibody against nqo1 - by Bioz Stars, 2020-04
    91/100 stars
      Buy from Supplier

    96
    Cell Signaling Technology Inc mouse anti nqo1
    KOK up-regulates Keap1-Nrf2 signaling in the SNpc and striatum following MPTP intoxication. (A) At 7 days after the last MPTP intoxication, SNpc and striatal sections ( n = 5 per group) were subjected to MitoSOX staining. The staining intensity was then quantified. Insets display high magnification micrographs of areas marked with squares. (B,C) Seven days after the last MPTP intoxication, SNpc, and striatum sample ( n = 3 per group) were performed by Western blot assay. Keap1, Nrf2, HO-1, and <t>NQO1.</t> SNpc (B) and striatum (C) . The top panels illustrate representative Western blots. ANOVA test; ## p
    Mouse Anti Nqo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti nqo1/product/Cell Signaling Technology Inc
    Average 96 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    mouse anti nqo1 - by Bioz Stars, 2020-04
    96/100 stars
      Buy from Supplier

    Image Search Results


    Association between NQO1 expression and β-lap cytotoxicity in melanoma cell lines. Levels of NQO1 expression determined by (A) western blotting and (B) viability following treatment with β-lap with or without simultaneous treatment with auxin in an AID-NQO1-expressing cell line. NQO1 expression levels in CRL-1585 cells determined by (C) western blotting, and (D) their viability following treatment with β-lap in comparison with cells treated with control siRNA or siRNA against NQO1. The results are presented as mean ± standard deviation from triplicate experiments (*P

    Journal: Oncology Letters

    Article Title: Carnosic acid, an inducer of NAD(P)H quinone oxidoreductase 1, enhances the cytotoxicity of β-lapachone in melanoma cell lines

    doi: 10.3892/ol.2017.7618

    Figure Lengend Snippet: Association between NQO1 expression and β-lap cytotoxicity in melanoma cell lines. Levels of NQO1 expression determined by (A) western blotting and (B) viability following treatment with β-lap with or without simultaneous treatment with auxin in an AID-NQO1-expressing cell line. NQO1 expression levels in CRL-1585 cells determined by (C) western blotting, and (D) their viability following treatment with β-lap in comparison with cells treated with control siRNA or siRNA against NQO1. The results are presented as mean ± standard deviation from triplicate experiments (*P

    Article Snippet: The antibody against NQO1 (cat. no. 3187) was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Expressing, Western Blot, Standard Deviation

    Carnosic acid stabilizes NRF2 and induces further expression of NQO1. Viability of (A) SK-MEL-28 and (B) GAK cells treated for 24 h with CA or SUL. One-way ANOVA followed by Tukey's honest significant difference test was used for statistical analysis (*P

    Journal: Oncology Letters

    Article Title: Carnosic acid, an inducer of NAD(P)H quinone oxidoreductase 1, enhances the cytotoxicity of β-lapachone in melanoma cell lines

    doi: 10.3892/ol.2017.7618

    Figure Lengend Snippet: Carnosic acid stabilizes NRF2 and induces further expression of NQO1. Viability of (A) SK-MEL-28 and (B) GAK cells treated for 24 h with CA or SUL. One-way ANOVA followed by Tukey's honest significant difference test was used for statistical analysis (*P

    Article Snippet: The antibody against NQO1 (cat. no. 3187) was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Expressing

    Effects of ISL on the antioxidant system and ROS in HepG2 cells. A. After various time treatments with ISL, confocal microscopy was used to visualize the intracellular colocalization of Keap-1 (green) and Nrf-2 (red). B. After various time treatments with ISL, expression of NQO1 and HO-1 proteins were measured by western blot. β-Actin was used as a standard. C. Intracellular ROS levels were monitored by using DCFH-DA staining, and fluorescence was analyzed using flow cytometry. All data are expressed as mean ± SEM from three independent experiments. * p

    Journal: American Journal of Cancer Research

    Article Title: Disturbance of redox status enhances radiosensitivity of hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: Effects of ISL on the antioxidant system and ROS in HepG2 cells. A. After various time treatments with ISL, confocal microscopy was used to visualize the intracellular colocalization of Keap-1 (green) and Nrf-2 (red). B. After various time treatments with ISL, expression of NQO1 and HO-1 proteins were measured by western blot. β-Actin was used as a standard. C. Intracellular ROS levels were monitored by using DCFH-DA staining, and fluorescence was analyzed using flow cytometry. All data are expressed as mean ± SEM from three independent experiments. * p

    Article Snippet: The membrane was blocked for 1 h in TBST containing 0.5% FBS and subsequently probed with anti-Nrf2 antibody (Santa Cruz, CA, USA), anti-Keap1 antibody (Santa Cruz), anti-HO1 antibody (Cell Signaling, Danvers, USA) and anti-NQO1 antibody (Cell Signaling) at 4°C overnight with shaking.

    Techniques: Confocal Microscopy, Expressing, Western Blot, Staining, Fluorescence, Flow Cytometry, Cytometry

    The effect of DADS on the protein expression of nuclear factor kappa B p65 (NF-κB p65), i-κB, nuclear factor erythroid 2-related factor 2 (Nrf-2), and NAD(P)H: quinone oxidoreductase 1 (NQO1) ( A ) Western blotting analyses of NF-κB p65, i-κB, Nrf-2, and NQO1 proteins; quantitative densitometric analyses of ( B ) NF-κB p65; ( C ) i-κB; ( D ) Nrf-2; and ( E ) NQO1 proteins normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each value represents the mean ± SEM of three independent experiments. ### p

    Journal: Nutrients

    Article Title: Pharmacological Investigation of the Anti-Inflammation and Anti-Oxidation Activities of Diallyl Disulfide in a Rat Emphysema Model Induced by Cigarette Smoke Extract

    doi: 10.3390/nu10010079

    Figure Lengend Snippet: The effect of DADS on the protein expression of nuclear factor kappa B p65 (NF-κB p65), i-κB, nuclear factor erythroid 2-related factor 2 (Nrf-2), and NAD(P)H: quinone oxidoreductase 1 (NQO1) ( A ) Western blotting analyses of NF-κB p65, i-κB, Nrf-2, and NQO1 proteins; quantitative densitometric analyses of ( B ) NF-κB p65; ( C ) i-κB; ( D ) Nrf-2; and ( E ) NQO1 proteins normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each value represents the mean ± SEM of three independent experiments. ### p

    Article Snippet: Anti-NF-κB p65, i-κB, and NQO1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Western Blot

    KOK up-regulates Keap1-Nrf2 signaling in the SNpc and striatum following MPTP intoxication. (A) At 7 days after the last MPTP intoxication, SNpc and striatal sections ( n = 5 per group) were subjected to MitoSOX staining. The staining intensity was then quantified. Insets display high magnification micrographs of areas marked with squares. (B,C) Seven days after the last MPTP intoxication, SNpc, and striatum sample ( n = 3 per group) were performed by Western blot assay. Keap1, Nrf2, HO-1, and NQO1. SNpc (B) and striatum (C) . The top panels illustrate representative Western blots. ANOVA test; ## p

    Journal: Frontiers in Pharmacology

    Article Title: Neuroprotective Effects of a Traditional Multi-Herbal Medicine Kyung-Ok-Ko in an Animal Model of Parkinson's Disease: Inhibition of MAPKs and NF-κB Pathways and Activation of Keap1-Nrf2 Pathway

    doi: 10.3389/fphar.2018.01444

    Figure Lengend Snippet: KOK up-regulates Keap1-Nrf2 signaling in the SNpc and striatum following MPTP intoxication. (A) At 7 days after the last MPTP intoxication, SNpc and striatal sections ( n = 5 per group) were subjected to MitoSOX staining. The staining intensity was then quantified. Insets display high magnification micrographs of areas marked with squares. (B,C) Seven days after the last MPTP intoxication, SNpc, and striatum sample ( n = 3 per group) were performed by Western blot assay. Keap1, Nrf2, HO-1, and NQO1. SNpc (B) and striatum (C) . The top panels illustrate representative Western blots. ANOVA test; ## p

    Article Snippet: For double immunofluorescent staining, sections were incubated overnight at 4°C with a mixture of mouse anti-PECAM-1 (1:500; Santa Cruz Biotechnology) and rabbit anti-GFAP (1:1,000; Millipore) antibodies or a mixture of rabbit anti-Nrf2 (1:500; Santa Cruz Biotechnology) and mouse anti-NQO1 (1:1,000; Cell Signaling Technology) antibodies.

    Techniques: Staining, Western Blot

    Nrf2 inhibitor neutralizes the protective effect of KOK following MPTP intoxication . (A–C) Nrf2 inhibitor (ML385; 30 mg/kg, i.p.) or saline was i.p. injected to mouse once daily from 30 min before KOK treatment in MPTP-intoxication model ( n = 4–6 per group). Pole test (A) , rotarod performance test (B) , and nest building behavior test (C) were accomplished at 5, 7, and 1 days, respectively, after the last MPTP intoxication. (D,E) Seven days after the last MPTP intoxication, nucleus, and cytosol isolated from SNpc (D) and striatum (E) from all groups ( n = 4 per group) were subjected to Western blot assay to quantify changes in Nrf2 nuclear translocation and NQO1 expression, respectively. (F) Seven days after the last MPTP intoxication, SNpc and striatal sections ( n = 3 per brain) were subjected to immunofluorescence staining using Nrf2 and NQO1 antibodies. Scale bar = 50 μm. ANOVA test; # p

    Journal: Frontiers in Pharmacology

    Article Title: Neuroprotective Effects of a Traditional Multi-Herbal Medicine Kyung-Ok-Ko in an Animal Model of Parkinson's Disease: Inhibition of MAPKs and NF-κB Pathways and Activation of Keap1-Nrf2 Pathway

    doi: 10.3389/fphar.2018.01444

    Figure Lengend Snippet: Nrf2 inhibitor neutralizes the protective effect of KOK following MPTP intoxication . (A–C) Nrf2 inhibitor (ML385; 30 mg/kg, i.p.) or saline was i.p. injected to mouse once daily from 30 min before KOK treatment in MPTP-intoxication model ( n = 4–6 per group). Pole test (A) , rotarod performance test (B) , and nest building behavior test (C) were accomplished at 5, 7, and 1 days, respectively, after the last MPTP intoxication. (D,E) Seven days after the last MPTP intoxication, nucleus, and cytosol isolated from SNpc (D) and striatum (E) from all groups ( n = 4 per group) were subjected to Western blot assay to quantify changes in Nrf2 nuclear translocation and NQO1 expression, respectively. (F) Seven days after the last MPTP intoxication, SNpc and striatal sections ( n = 3 per brain) were subjected to immunofluorescence staining using Nrf2 and NQO1 antibodies. Scale bar = 50 μm. ANOVA test; # p

    Article Snippet: For double immunofluorescent staining, sections were incubated overnight at 4°C with a mixture of mouse anti-PECAM-1 (1:500; Santa Cruz Biotechnology) and rabbit anti-GFAP (1:1,000; Millipore) antibodies or a mixture of rabbit anti-Nrf2 (1:500; Santa Cruz Biotechnology) and mouse anti-NQO1 (1:1,000; Cell Signaling Technology) antibodies.

    Techniques: Injection, Isolation, Western Blot, Translocation Assay, Expressing, Immunofluorescence, Staining