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elisa-based transam nrf2 kit  (Active Motif)
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Active Motif elisa-based transam nrf2 kit
(A and B) Semiconfluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours; pretreated with vehicle or sulforaphane (SFN) (2–4 μM) for 1 hour; and then incubated with serum-free medium or medium containing 2.5% fetal bovine serum (FBS). DNA synthesis was determined by measuring bromodeoxyuridine (BrdU) incorporation 48 hours after treatment (A). p21Waf1and p27Kip1 expression was determined 24 hours after treatment in whole-cell protein extracts by Western blotting and normalized to β-actin expression (B). (C and D) ASMCs were incubated with adenoviral vectors expressing GFP (Ad-GFP) and wild-type nuclear factor E2-related factor 2 <t>(Ad-Nrf2)</t> (multiplicity of infection 250) for 18 hours, serum-deprived for 6 hours, and then incubated with serum-free medium or medium containing 2.5% FBS. DNA synthesis was determined by measuring BrdU incorporation 72 hours after treatment (C). p21Waf1and p27Kip1 expression was determined 72 hours after treatment in whole-cell protein extracts by Western blotting and normalized to β-actin expression (D). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle control or Ad-GFP. Bars represent mean ± SEM of six ASMC (A and C) and four ASMC donors (B and D).
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(A and B) Semiconfluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours; pretreated with vehicle or sulforaphane (SFN) (2–4 μM) for 1 hour; and then incubated with serum-free medium or medium containing 2.5% fetal bovine serum (FBS). DNA synthesis was determined by measuring bromodeoxyuridine (BrdU) incorporation 48 hours after treatment (A). p21Waf1and p27Kip1 expression was determined 24 hours after treatment in whole-cell protein extracts by Western blotting and normalized to β-actin expression (B). (C and D) ASMCs were incubated with adenoviral vectors expressing GFP (Ad-GFP) and wild-type nuclear factor E2-related factor 2 (Ad-Nrf2) (multiplicity of infection 250) for 18 hours, serum-deprived for 6 hours, and then incubated with serum-free medium or medium containing 2.5% FBS. DNA synthesis was determined by measuring BrdU incorporation 72 hours after treatment (C). p21Waf1and p27Kip1 expression was determined 72 hours after treatment in whole-cell protein extracts by Western blotting and normalized to β-actin expression (D). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle control or Ad-GFP. Bars represent mean ± SEM of six ASMC (A and C) and four ASMC donors (B and D).

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Transforming Growth Factor-? and Nuclear Factor E2-related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells

doi: 10.1164/rccm.201011-1780OC

Figure Lengend Snippet: (A and B) Semiconfluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours; pretreated with vehicle or sulforaphane (SFN) (2–4 μM) for 1 hour; and then incubated with serum-free medium or medium containing 2.5% fetal bovine serum (FBS). DNA synthesis was determined by measuring bromodeoxyuridine (BrdU) incorporation 48 hours after treatment (A). p21Waf1and p27Kip1 expression was determined 24 hours after treatment in whole-cell protein extracts by Western blotting and normalized to β-actin expression (B). (C and D) ASMCs were incubated with adenoviral vectors expressing GFP (Ad-GFP) and wild-type nuclear factor E2-related factor 2 (Ad-Nrf2) (multiplicity of infection 250) for 18 hours, serum-deprived for 6 hours, and then incubated with serum-free medium or medium containing 2.5% FBS. DNA synthesis was determined by measuring BrdU incorporation 72 hours after treatment (C). p21Waf1and p27Kip1 expression was determined 72 hours after treatment in whole-cell protein extracts by Western blotting and normalized to β-actin expression (D). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle control or Ad-GFP. Bars represent mean ± SEM of six ASMC (A and C) and four ASMC donors (B and D).

Article Snippet: Nrf2-ARE Binding Assay Nrf2-ARE binding was determined using an ELISA-based TransAM Nrf2 Kit (Active Motif, Rixensart, Belgium) according to the manufacturer's instructions.

Techniques: Incubation, DNA Synthesis, BrdU Incorporation Assay, Expressing, Western Blot, Infection

(A and B) Confluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours and then treated with transforming growth factor (TGF)-β (1 ng/ml) for 0.5–24 hours (A), or TGF-β (0.25–1 ng/ml) for 24 hours (B). Heme oxygenase (HO)-1 and NAD(P)H:quinone oxidoreductase (NQO1) mRNA was determined by real-time polymerase chain reaction (PCR) and normalized to 18S rRNA expression. (C and D) Confluent ASMCs were transfected with antioxidant response elements (ARE)–driven luciferase reporter vector for 18 hours, serum-deprived for 6 hours, and finally treated with TGF-β (0.25–1 ng/ml) for 20 hours (C) or pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour and then treated with TGF-β (0.25 ng/ml) for 20 hours (D). ARE-driven transcriptional activity was determined by measuring firefly luciferase activity and normalizing to Renilla luciferase activity. (E and F) Confluent ASMCs were serum-deprived for 24 hours and then treated with TGF-β (0.25–1 ng/ml) for 20 hours. Nuclear factor E2-related factor 2 (Nrf2) expression was determined in whole-cell extracts by Western blotting and normalized to β-actin expression (E). Nrf2-ARE binding was determined in nuclear extracts by an ELISA-based TransAM assay (F). Bars represent mean ± SEM of three ASMC (A and F), five ASMC (B), three to six ASMC (C), four ASMC (D), and four ASMC donors (E). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with unstimulated control. ns = nonsignificant.

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Transforming Growth Factor-? and Nuclear Factor E2-related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells

doi: 10.1164/rccm.201011-1780OC

Figure Lengend Snippet: (A and B) Confluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours and then treated with transforming growth factor (TGF)-β (1 ng/ml) for 0.5–24 hours (A), or TGF-β (0.25–1 ng/ml) for 24 hours (B). Heme oxygenase (HO)-1 and NAD(P)H:quinone oxidoreductase (NQO1) mRNA was determined by real-time polymerase chain reaction (PCR) and normalized to 18S rRNA expression. (C and D) Confluent ASMCs were transfected with antioxidant response elements (ARE)–driven luciferase reporter vector for 18 hours, serum-deprived for 6 hours, and finally treated with TGF-β (0.25–1 ng/ml) for 20 hours (C) or pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour and then treated with TGF-β (0.25 ng/ml) for 20 hours (D). ARE-driven transcriptional activity was determined by measuring firefly luciferase activity and normalizing to Renilla luciferase activity. (E and F) Confluent ASMCs were serum-deprived for 24 hours and then treated with TGF-β (0.25–1 ng/ml) for 20 hours. Nuclear factor E2-related factor 2 (Nrf2) expression was determined in whole-cell extracts by Western blotting and normalized to β-actin expression (E). Nrf2-ARE binding was determined in nuclear extracts by an ELISA-based TransAM assay (F). Bars represent mean ± SEM of three ASMC (A and F), five ASMC (B), three to six ASMC (C), four ASMC (D), and four ASMC donors (E). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with unstimulated control. ns = nonsignificant.

Article Snippet: Nrf2-ARE Binding Assay Nrf2-ARE binding was determined using an ELISA-based TransAM Nrf2 Kit (Active Motif, Rixensart, Belgium) according to the manufacturer's instructions.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

Confluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours, pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour, and then stimulated with transforming growth factor (TGF)-β (0.25 ng/ml) for 24 hours (A–C). Alternatively, ASMCs were incubated with adenoviral vectors expressing GFP (Ad-GFP) and wild-type nuclear factor E2-related factor 2 (Ad-Nrf2) (multiplicity of infection 250) for 18 hours, serum-deprived for 24 hours, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours (C–E). Heme oxygenase (HO)-1, catalase, and manganese superoxide dismutase (MnSOD) mRNA expression was determined by real-time polymerase chain reaction and normalized to 18S rRNA expression. Bars represent mean ± SEM of five ASMC (A–C) and four ASMC donors (C–E). *P < 0.05, **P < 0.01. ns = non-significant.

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Transforming Growth Factor-? and Nuclear Factor E2-related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells

doi: 10.1164/rccm.201011-1780OC

Figure Lengend Snippet: Confluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours, pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour, and then stimulated with transforming growth factor (TGF)-β (0.25 ng/ml) for 24 hours (A–C). Alternatively, ASMCs were incubated with adenoviral vectors expressing GFP (Ad-GFP) and wild-type nuclear factor E2-related factor 2 (Ad-Nrf2) (multiplicity of infection 250) for 18 hours, serum-deprived for 24 hours, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours (C–E). Heme oxygenase (HO)-1, catalase, and manganese superoxide dismutase (MnSOD) mRNA expression was determined by real-time polymerase chain reaction and normalized to 18S rRNA expression. Bars represent mean ± SEM of five ASMC (A–C) and four ASMC donors (C–E). *P < 0.05, **P < 0.01. ns = non-significant.

Article Snippet: Nrf2-ARE Binding Assay Nrf2-ARE binding was determined using an ELISA-based TransAM Nrf2 Kit (Active Motif, Rixensart, Belgium) according to the manufacturer's instructions.

Techniques: Incubation, Expressing, Infection, Real-time Polymerase Chain Reaction

(A and B) Semiconfluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours, pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour, and then incubated with medium containing 2.5% fetal bovine serum (FBS) or 2.5% FBS and transforming growth factor (TGF)-β (0.25 ng/ml) for 72 hours (A). Alternatively, ASMCs were incubated with adenoviral vectors expressing GFP (Ad-GFP) and wild-type nuclear factor E2-related factor 2 (Ad-Nrf2) (multiplicity of infection 250) for 18 hours, serum-deprived for 6 hours, and then incubated with medium containing 2.5% FBS or 2.5% FBS and TGF-β (0.25 ng/ml) for 72 hours (B). DNA synthesis was determined by measuring bromodeoxyuridine (BrdU) incorporation. (C–F) Confluent ASMCs were serum-deprived for 24 hours, pretreated with vehicle control or sulforaphane (2–4 μM) for 1 hour, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours (C and D). Alternatively, ASMCs were incubated with Ad-GFP or Ad-Nrf2 (MOI 250) for 18 hours, serum-deprived for 6 hours, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours (E and F). IL-6 mRNA expression was determined by real-time polymerase chain reaction, normalized to 18S rRNA expression, and expressed as fold change with respect to unstimulated control. IL-6 release was determined by ELISA. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle or Ad-GFP control. #P < 0.05, ##P < 0.01, and ###P < 0.001 compared with TGF-β and vehicle or Ad-GFP–treated cells. Bars represent mean ± SEM of five ASMC donors (B and C), four ASMC donors (A, D, and F), and three ASMC donors (E).

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Transforming Growth Factor-? and Nuclear Factor E2-related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells

doi: 10.1164/rccm.201011-1780OC

Figure Lengend Snippet: (A and B) Semiconfluent airway smooth muscle cells (ASMCs) were serum-deprived for 24 hours, pretreated with vehicle or sulforaphane (2–4 μM) for 1 hour, and then incubated with medium containing 2.5% fetal bovine serum (FBS) or 2.5% FBS and transforming growth factor (TGF)-β (0.25 ng/ml) for 72 hours (A). Alternatively, ASMCs were incubated with adenoviral vectors expressing GFP (Ad-GFP) and wild-type nuclear factor E2-related factor 2 (Ad-Nrf2) (multiplicity of infection 250) for 18 hours, serum-deprived for 6 hours, and then incubated with medium containing 2.5% FBS or 2.5% FBS and TGF-β (0.25 ng/ml) for 72 hours (B). DNA synthesis was determined by measuring bromodeoxyuridine (BrdU) incorporation. (C–F) Confluent ASMCs were serum-deprived for 24 hours, pretreated with vehicle control or sulforaphane (2–4 μM) for 1 hour, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours (C and D). Alternatively, ASMCs were incubated with Ad-GFP or Ad-Nrf2 (MOI 250) for 18 hours, serum-deprived for 6 hours, and then stimulated with TGF-β (0.25 ng/ml) for 24 hours (E and F). IL-6 mRNA expression was determined by real-time polymerase chain reaction, normalized to 18S rRNA expression, and expressed as fold change with respect to unstimulated control. IL-6 release was determined by ELISA. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle or Ad-GFP control. #P < 0.05, ##P < 0.01, and ###P < 0.001 compared with TGF-β and vehicle or Ad-GFP–treated cells. Bars represent mean ± SEM of five ASMC donors (B and C), four ASMC donors (A, D, and F), and three ASMC donors (E).

Article Snippet: Nrf2-ARE Binding Assay Nrf2-ARE binding was determined using an ELISA-based TransAM Nrf2 Kit (Active Motif, Rixensart, Belgium) according to the manufacturer's instructions.

Techniques: Incubation, Expressing, Infection, DNA Synthesis, BrdU Incorporation Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Airway smooth muscle cells (ASMCs) cultured from bronchoscopic biopsies and transplant airways taken from healthy subjects (n = 5–6) or bronchoscopic biopsies from patients with nonsevere (n = 6) and severe asthma (n = 6–7) were grown to confluence in medium containing 10% fetal bovine serum, and whole-cell protein was extracted. (A and B) Nuclear factor E2-related factor 2 (Nrf2) protein expression was determined in whole-cell protein extracts by Western blotting and normalized to β-actin expression. To ensure that all membranes were equally exposed to antibodies, substrate, and X-ray film a control sample (c) was run in each of the gels. (C) Nrf2–antioxidant response elements (ARE) binding was determined in whole-cell protein extracts by an ELISA-based TransAM assay. Data were analyzed using Mann-Whitney test.

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Transforming Growth Factor-? and Nuclear Factor E2-related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells

doi: 10.1164/rccm.201011-1780OC

Figure Lengend Snippet: Airway smooth muscle cells (ASMCs) cultured from bronchoscopic biopsies and transplant airways taken from healthy subjects (n = 5–6) or bronchoscopic biopsies from patients with nonsevere (n = 6) and severe asthma (n = 6–7) were grown to confluence in medium containing 10% fetal bovine serum, and whole-cell protein was extracted. (A and B) Nuclear factor E2-related factor 2 (Nrf2) protein expression was determined in whole-cell protein extracts by Western blotting and normalized to β-actin expression. To ensure that all membranes were equally exposed to antibodies, substrate, and X-ray film a control sample (c) was run in each of the gels. (C) Nrf2–antioxidant response elements (ARE) binding was determined in whole-cell protein extracts by an ELISA-based TransAM assay. Data were analyzed using Mann-Whitney test.

Article Snippet: Nrf2-ARE Binding Assay Nrf2-ARE binding was determined using an ELISA-based TransAM Nrf2 Kit (Active Motif, Rixensart, Belgium) according to the manufacturer's instructions.

Techniques: Cell Culture, Expressing, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY