c terminal nrf2 antibody  (R&D Systems)

Journal: Cells

Article Title: Switching of Redox Signaling by Prdx6 Expression Decides Cellular Fate by Hormetic Phenomena Involving Nrf2 and Reactive Oxygen Species

doi: 10.3390/cells11081266

Figure Lengend Snippet: Aberrant Nrf2 and Klf9 expression by excessive oxidative stress was associated with reduced Prdx6 repression, ROS accumulation and increased cell death. ( A ) Cellular and nuclear extract were prepared from hLECs exposed to increasing doses of H 2 O 2 as indicated in Figure. Samples containing equal amount of protein were immunoblotted using antibody specific to Nrf2 or Klf9 or LaminB1 or Prdx6 or β-actin as shown, and an increased nuclear accumulation of Nrf2 and Klf9 ( Aa ) and reduced in Prdx6 expression ( Ab ) were observed in response to the increased concentrations of H 2 O 2 treatment. Right adjacent panel reveals densitometry analysis of each protein bands. ( B ) hLECs were exposed to different concentrations of H 2 O 2 . 3 h later total RNA was isolated. Real-time qPCR analysis of Klf9 ( Ba ) and Prdx6 ( Bb ) with their corresponding specific primers. Data are the mean ± S.D. value of three independent experiments. * p < 0.001 versus control. ( C ) H 2 O 2 treated hLECs showed a significant increase in Nrf2 activity. hLECs were treated with different concentrations of H 2 O 2 for 3 h, and nuclear extract were analyzed for Nrf2-ARE (antioxidant responsive element) binding by ELISA (enzyme-linked immunosorbent assay). Nuclear extracts containing equal amount of protein were processed and assayed for Nrf2 activity with a commercially available kit (Active motif) as described in Materials and Methods. The data represent the mean ± S.D. from three independent experiments. p values were determined; H 2 O 2 treated versus untreated control. * p < 0.001. Note: to avoid effect of cell death against excessive oxidative stress, cell extracts were prepared at 3 h of oxidant exposure for the study. ( D ) Cultured hLECs treated with increasing concentrations of H 2 O 2 , 3 h later ROS were measured by H2-DCF-DH dye and data were presented as DCF oxidation levels. Data are the mean ± S.D. of three independent experiments. * p < 0.001 vs. control. ( E ) MTS assay was conducted to examine cell viability against oxidative stress after 24 h, and was well corelated to ROS levels. Data are the mean ± S.D. of three independent experiments. * p < 0.001 vs. control.

Article Snippet: Antibodies used were control IgG, an antibody specific to Klf9 (Catalog No. 701888, Thermo Scientific) and Nrf2 (Catalog No. Ab180845, Abcam).

Techniques: Expressing, Isolation, Control, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, MTS Assay

Journal: Cells

Article Title: Switching of Redox Signaling by Prdx6 Expression Decides Cellular Fate by Hormetic Phenomena Involving Nrf2 and Reactive Oxygen Species

doi: 10.3390/cells11081266

Figure Lengend Snippet: Nrf2 binding was increased to ARE sites present in Klf9 promoter in response to excessive oxidative stress. ( A ) Top panel displaying the presence of four ARE sites in Klf9 promoter and ChIP-qPCR primers consisting of ARE3 and ARE4. ( B ) In vivo DNA binding assay disclosed Nrf2/ARE interaction. hLECs treated with different concentrations of H 2 O 2 (150 and 300 µM) for 3 h. ChIP assay was carried out with Nrf2 and control IgG antibodies. Pulled DNA fragments were subjected to RT-qPCR analysis. DNA fragments present in the immunoprecipitation were amplified with primers that specifically recognized a fragment of Klf9 promoter containing ARE site three and ARE site four as indicated. 10% chromatin was used as input. As a negative control, ChIP with control IgG was used. # and ## denotes primer pairs used for ChIP-RT-PCR.

Article Snippet: Antibodies used were control IgG, an antibody specific to Klf9 (Catalog No. 701888, Thermo Scientific) and Nrf2 (Catalog No. Ab180845, Abcam).

Techniques: Binding Assay, ChIP-qPCR, In Vivo, DNA Binding Assay, Control, Quantitative RT-PCR, Immunoprecipitation, Amplification, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Cells

Article Title: Switching of Redox Signaling by Prdx6 Expression Decides Cellular Fate by Hormetic Phenomena Involving Nrf2 and Reactive Oxygen Species

doi: 10.3390/cells11081266

Figure Lengend Snippet: Actinomycin D treatment revealed that Klf9 induction by oxidative stress did not occur post-transcriptionally. ( A , B ) hLECs were pretreated with Actinomycin D (Act D) for 30 min followed by H 2 O 2 for 3 h and proceeded for protein ( A ) and mRNA ( B ) analysis with Klf9 ( Ba ) or Nrf2 ( Bb ) specific probes as indicated. Data are the mean ± S.D. of three independent experiments. * p < 0.001 vs. control.

Article Snippet: Antibodies used were control IgG, an antibody specific to Klf9 (Catalog No. 701888, Thermo Scientific) and Nrf2 (Catalog No. Ab180845, Abcam).

Techniques: Control

Journal: Cells

Article Title: Switching of Redox Signaling by Prdx6 Expression Decides Cellular Fate by Hormetic Phenomena Involving Nrf2 and Reactive Oxygen Species

doi: 10.3390/cells11081266

Figure Lengend Snippet: Loss and gain experiments of Nrf2 disclosed that altered Klf9 expression and its promoter activity was directly linked to cellular levels of Nrf2. ( A , B ) Nrf2 -deficient hLECs fails to activate the Klf9 expression and its transcription. ( A ) Nrf2 -depleted hLECs were exposed to H 2 O 2 as indicated in A for 3 h. Nuclear extract were prepared, and equal amount of protein was immunoblotted using an antibody specific to Nrf2 or Klf9. ( B ) Nrf2 -deficeint hLECs were transfected with WT Klf9 promoter (−5856/+71) linked to CAT vector. The transfectants were exposed to H 2 O 2 for 3 h as shown, and Klf9 promoter activity was measured and shown. Data are the mean ± S.D. of three independent experiments. * p < 0.001; H 2 O 2 treated versus control and Sh -Nrf2 vs. Sh -Control. ( C – E ) Nrf2 overexpression enhanced the Klf9 promoter activity. ( C ) Photomicrograph representing the Nrf2 overexpression in hLECs, left panel: pEGFP-Vector; right panel: pEGFP-Nrf2. ( D ) Western blot analysis showing the Nrf2 overexpression of transfected cells. Cellular extract prepared from pEGFP-Vector and pEGFP-Nrf2 overexpressed hLECs having equal amount of protein was immunoblotted with antibody specific to Nrf2. ( E ) Cells overexpressing Nrf2 showed enhanced Klf9 transcription. hLECs were transfected with Klf9 promoter (−5856/+71) along with pEGFP-vector and/or pEGFP-Nrf2 plasmids. 48 h later transfectants were exposed to H 2 O 2 for 3 h, and promoter activity was monitored. Data are the mean ± S.D. of three independent experiments. * p < 0.001; H 2 O 2 treated versus control and pEGFP-Nrf2 vs. pEGFP-Vector.

Article Snippet: Antibodies used were control IgG, an antibody specific to Klf9 (Catalog No. 701888, Thermo Scientific) and Nrf2 (Catalog No. Ab180845, Abcam).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Control, Over Expression, Western Blot

Journal: Cells

Article Title: Switching of Redox Signaling by Prdx6 Expression Decides Cellular Fate by Hormetic Phenomena Involving Nrf2 and Reactive Oxygen Species

doi: 10.3390/cells11081266

Figure Lengend Snippet: Diagrammatic Illustration of the molecular mechanism governing the bimodal effect of ROS responsible for cellular response in response to oxidative stress. Mild to moderate oxidative stress regulates the Nrf2 at physiological level while excessive oxidative stress aberrantly accumulates the Nrf2 into nucleus. Physiological regulations of Nrf2 activates its target antioxidant genes transcription and expression by binding to antioxidant response element (ARE) sites present in the regulatory region of antioxidant genes and maintain the cellular ROS homeostasis which leads to beneficial cell signaling. At higher levels of oxidative stress, aberrant accumulations of Nrf2 in nucleus, paradoxically activated Klf9 transcription by binding to its ARE sites present in Klf9 gene promoter, and this aberrant Klf9 expression repressed some of the antioxidant genes, such as Prdx6. Finally, these phenomena result in Klf9 abundance-dependent increased ROS amplification, and subsequent cell injuries/death at least in part by transcriptional suppression of Prdx6. Taken together, our data demonstrate that Nrf2-Klf9-Prdx6-axis can play a role in determining ROS-mediated signaling pathway and biological response(s) in conditions of oxidative stress.

Article Snippet: Antibodies used were control IgG, an antibody specific to Klf9 (Catalog No. 701888, Thermo Scientific) and Nrf2 (Catalog No. Ab180845, Abcam).

Techniques: Expressing, Binding Assay, Amplification

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 2. Keap1 protein decreases in differentiated adipocytes, while steady-state mRNA expression for Nrf2 and Keap1 increases. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 0, 4, 8, 11, and 14. (A) Nrf2 and (C) Keap1 (70 kDa) protein expression was examined by immunoblotting. Black bars, full-length (FL) Nrf2 (56 kDa); gray bars, degraded (Deg) Nrf2 (40 kDa); white bars, total (Tot) Nrf2; the sums of full-length Nrf2 and degraded Nrf2 were determined to obtain relative total Nrf2 protein expression. (B) Relative steady-state Nrf2 and (D) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 0, bars indicate Pr0.05 between samples. Data are expressed as mean7SD; n¼3–4.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Expressing, Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 3. NQO1 protein increases during limited clonal expansion and postmitotic growth arrest of differentiation. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 0, 1, 2, 3, and 4. (A) NQO1 and (D) Keap1 protein expressions were examined by immunoblotting. (B) Relative NQO1, (C) Nrf2, and (E) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 0, bars indicate Pr0.05 between samples. Data are expressed as mean7SD; n¼3–4.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 4. NQO1 and Keap1 proteins, but not mRNA, decreases as adipocyte differentiation progresses. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 4, 5, 6, 7, and 8. (A) NQO1 and (D) Keap1 protein expressions were examined by immunoblotting. (B) Relative NQO1, (C) Nrf2, and (E) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 4. Data are expressed as mean7SD; n¼3.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 6. LiCl treatment increases NQO1 protein, but not NQO1 mRNA. 3T3-L1 cells were differentiated as described under Materials and methods, and treated with varying concentrations of LiCl at Day 8 and harvested on Day 11. A 40 mM NaCl was used as a vehicle control (VC). (A) NQO1 was examined by immunoblotting. (B) Relative NQO1 and (C) Nrf2 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s; * Pr0.05 as compared to VC for protein and mRNA. Data are expressed as mean7SD; n¼3.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Control, Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 7. Sulforaphane increases NQO1 protein and mRNA levels. 3T3-L1 cells were differentiated as described under Materials and methods, and treated with varying concentrations of sulforaphane (SFN). A 0.3% DMSO was used as a VC. (A) NQO1 was examined by immunoblotting. (B) Relative NQO1 and (C) Nrf2 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s; * Pr0.05 as compared to VC for protein and mRNA. Data are expressed as mean7SD; n¼3.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 2. Keap1 protein decreases in differentiated adipocytes, while steady-state mRNA expression for Nrf2 and Keap1 increases. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 0, 4, 8, 11, and 14. (A) Nrf2 and (C) Keap1 (70 kDa) protein expression was examined by immunoblotting. Black bars, full-length (FL) Nrf2 (56 kDa); gray bars, degraded (Deg) Nrf2 (40 kDa); white bars, total (Tot) Nrf2; the sums of full-length Nrf2 and degraded Nrf2 were determined to obtain relative total Nrf2 protein expression. (B) Relative steady-state Nrf2 and (D) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 0, bars indicate Pr0.05 between samples. Data are expressed as mean7SD; n¼3–4.

Article Snippet: When Nrf2 protein was assessed by immunoblot, there was an immunoreactive protein signal for Nrf2 at approximately 56 kDa and a smaller immunoreactive protein signal at 40 kDa of putatively degraded or possibly an isoform of Nrf2 protein when the Santa Cruz C-terminal Nrf2 antibody (No. sc-722) was used.

Techniques: Expressing, Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 3. NQO1 protein increases during limited clonal expansion and postmitotic growth arrest of differentiation. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 0, 1, 2, 3, and 4. (A) NQO1 and (D) Keap1 protein expressions were examined by immunoblotting. (B) Relative NQO1, (C) Nrf2, and (E) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 0, bars indicate Pr0.05 between samples. Data are expressed as mean7SD; n¼3–4.

Article Snippet: When Nrf2 protein was assessed by immunoblot, there was an immunoreactive protein signal for Nrf2 at approximately 56 kDa and a smaller immunoreactive protein signal at 40 kDa of putatively degraded or possibly an isoform of Nrf2 protein when the Santa Cruz C-terminal Nrf2 antibody (No. sc-722) was used.

Techniques: Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 4. NQO1 and Keap1 proteins, but not mRNA, decreases as adipocyte differentiation progresses. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 4, 5, 6, 7, and 8. (A) NQO1 and (D) Keap1 protein expressions were examined by immunoblotting. (B) Relative NQO1, (C) Nrf2, and (E) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 4. Data are expressed as mean7SD; n¼3.

Article Snippet: When Nrf2 protein was assessed by immunoblot, there was an immunoreactive protein signal for Nrf2 at approximately 56 kDa and a smaller immunoreactive protein signal at 40 kDa of putatively degraded or possibly an isoform of Nrf2 protein when the Santa Cruz C-terminal Nrf2 antibody (No. sc-722) was used.

Techniques: Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 6. LiCl treatment increases NQO1 protein, but not NQO1 mRNA. 3T3-L1 cells were differentiated as described under Materials and methods, and treated with varying concentrations of LiCl at Day 8 and harvested on Day 11. A 40 mM NaCl was used as a vehicle control (VC). (A) NQO1 was examined by immunoblotting. (B) Relative NQO1 and (C) Nrf2 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s; * Pr0.05 as compared to VC for protein and mRNA. Data are expressed as mean7SD; n¼3.

Article Snippet: When Nrf2 protein was assessed by immunoblot, there was an immunoreactive protein signal for Nrf2 at approximately 56 kDa and a smaller immunoreactive protein signal at 40 kDa of putatively degraded or possibly an isoform of Nrf2 protein when the Santa Cruz C-terminal Nrf2 antibody (No. sc-722) was used.

Techniques: Control, Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 7. Sulforaphane increases NQO1 protein and mRNA levels. 3T3-L1 cells were differentiated as described under Materials and methods, and treated with varying concentrations of sulforaphane (SFN). A 0.3% DMSO was used as a VC. (A) NQO1 was examined by immunoblotting. (B) Relative NQO1 and (C) Nrf2 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s; * Pr0.05 as compared to VC for protein and mRNA. Data are expressed as mean7SD; n¼3.

Article Snippet: When Nrf2 protein was assessed by immunoblot, there was an immunoreactive protein signal for Nrf2 at approximately 56 kDa and a smaller immunoreactive protein signal at 40 kDa of putatively degraded or possibly an isoform of Nrf2 protein when the Santa Cruz C-terminal Nrf2 antibody (No. sc-722) was used.

Techniques: Western Blot