recombinant nrf2 rnrf2  (Novus Biologicals)

Journal: Antioxidants

Article Title: NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus

doi: 10.3390/antiox11081582

Figure Lengend Snippet: CSE promoter activity in response to RSV infection, tBHQ stimulation and NRF2 expression. iSAE cells were transfected with -973 CSE-PGL4-Luc and Renilla-Luc plasmid (pRL-CMV) in 24-well plates. After 24 h, transfected cells were left untreated (control) or treated with tBHQ (25 µM) for 15 h or infected with RSV for 24 h alone or in combination with tBHQ (RSV + tBHQ). Luciferase activity was normalized to Renilla luciferase activity. Results are expressed as mean ± SEM of triplicate determination of a representative experiment repeated twice and expressed as fold change relative to control. * p < 0.05 relative to control cells, # p < 0.05 compared to RSV infected cells ( A ). iSAE cells were co-transfected with increasing amounts of NRF2 expression vector or the corresponding empty vector and harvested 48 h post transfection to measure luciferase activity. Luciferase activity was normalized to Renilla luciferase activity. Results are expressed as mean ± SEM of triplicate determination of a representative experiment repeated twice and expressed as fold change relative to control. * p < 0.05 relative to empty vector ( B ). Each circle represents individual data.

Article Snippet: The sequences of oligonucleotides are shown: −156/−120 CSE wild type (WT) 5′-CGGTCGCGGTGACGTTTCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCCACTGCAAAGTCCGTTGCGGAGACGACA-5′ −156/−120 CSE mutant (M) 5′-CGGTCGCGG GT C AG T G TCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCC CA G TC A C AGTCCGTTGCGGAGACGACA-5′ Commercially available purified human recombinant NRF2 (rNRF2) (Novus Biologicals, Littleton, CO, USA) was used as a positive control.

Techniques: Activity Assay, Infection, Expressing, Transfection, Plasmid Preparation, Control, Luciferase

Journal: Antioxidants

Article Title: NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus

doi: 10.3390/antiox11081582

Figure Lengend Snippet: Effects of 5’ deletions in the CSE promoter sequence on NRF2-inducible activity. Schematic representation of the CSE promoter deletion constructs. Numbering is relative to transcription initiation site (+1) ( A ). iSAE cells were co-transfected with the CSE-PGL4-Luc deletion mutants and Renilla-Luc plasmid plus either the empty vector or the NRF2 expression vector and harvested 48 h later to measure luciferase activity. Luciferase activity was normalized to Renilla luciferase activity. Results are expressed as mean ± SEM of triplicate determination of a representative experiment repeated twice and expressed as fold change relative to empty vector. * p < 0.05 relative to −973-PGL4-Luc ( B ). iSAE cells were co-transfected with the CSE-PGL4-Luc deletion mutants and Renilla-Luc plasmid for 24 h. Cells were left untreated or treated with tBHQ (25 µM) for 15 h before harvesting to measure luciferase activity. Luciferase activity was normalized to Renilla luciferase activity. Results are expressed as mean ± SEM of triplicate determination of a representative experiment repeated twice and expressed as fold change relative to control. * p < 0.05 relative to control cells ( C ). Each circle represents individual data.

Article Snippet: The sequences of oligonucleotides are shown: −156/−120 CSE wild type (WT) 5′-CGGTCGCGGTGACGTTTCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCCACTGCAAAGTCCGTTGCGGAGACGACA-5′ −156/−120 CSE mutant (M) 5′-CGGTCGCGG GT C AG T G TCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCC CA G TC A C AGTCCGTTGCGGAGACGACA-5′ Commercially available purified human recombinant NRF2 (rNRF2) (Novus Biologicals, Littleton, CO, USA) was used as a positive control.

Techniques: Sequencing, Activity Assay, Construct, Transfection, Plasmid Preparation, Expressing, Luciferase, Control

Journal: Antioxidants

Article Title: NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus

doi: 10.3390/antiox11081582

Figure Lengend Snippet: Effects of site mutations in the CSE promoter sequence on NRF2-inducible activity. Schematic representation of the CSE promoter site mutation constructs. Numbering is relative to transcription initiation site. Four segments of nine nucleotides for each of the 36 base pair regions of the CSE promoter comprised between nt −156 and −120 were mutated separately (mutation sites shown in italic underlined) ( A ). iSAE cells were co-transfected with the wild type (WT) −250-CSE-PGL4-Luc or the site mutants (M1 to M4) and Renilla-Luc plasmid plus either the empty vector or the NRF2 expression vector and harvested 48 h later to measure luciferase activity. Luciferase activity was normalized to Renilla luciferase activity. Results are expressed as mean ± SEM of triplicate determination of a representative experiment repeated twice and expressed as fold change relative to empty vector (each circle represents individual data). * p < 0.05 relative to WT PGL4-Luc ( B ). Nucleotide comparison of putative CSE antioxidant response element (ARE) with canonical (NQO1) and non-canonical (heme oxigenase-1, HO-1 and HSF1) ARE sites ( C ).

Article Snippet: The sequences of oligonucleotides are shown: −156/−120 CSE wild type (WT) 5′-CGGTCGCGGTGACGTTTCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCCACTGCAAAGTCCGTTGCGGAGACGACA-5′ −156/−120 CSE mutant (M) 5′-CGGTCGCGG GT C AG T G TCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCC CA G TC A C AGTCCGTTGCGGAGACGACA-5′ Commercially available purified human recombinant NRF2 (rNRF2) (Novus Biologicals, Littleton, CO, USA) was used as a positive control.

Techniques: Sequencing, Activity Assay, Mutagenesis, Construct, Transfection, Plasmid Preparation, Expressing, Luciferase, Comparison

Journal: Antioxidants

Article Title: NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus

doi: 10.3390/antiox11081582

Figure Lengend Snippet: EMSA of CSE putative ARE binding complexes in response to tBHQ treatment. Nuclear extracts (6 µg) from control and SAE cells treated with tBHQ (25 µM) for 15 h were used in EMSA. Two DNA–protein complexes, C1 and C2, are detected in control cells. C2 binding is further increased by tBHQ treatment ( A ). Similar complex formation is observed with purified recombinant NRF2 (rNRF2) ( B ). Competition assay using wild type (WT) and mutant (M) oligos (50×) and nuclear extracts of tBHQ-treated SAE cells ( C ). Super-shift assay with specific anti-NRF2 antibody and control IgG using nuclear extracts of tBHQ-treated SAE cells ( D ).

Article Snippet: The sequences of oligonucleotides are shown: −156/−120 CSE wild type (WT) 5′-CGGTCGCGGTGACGTTTCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCCACTGCAAAGTCCGTTGCGGAGACGACA-5′ −156/−120 CSE mutant (M) 5′-CGGTCGCGG GT C AG T G TCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCC CA G TC A C AGTCCGTTGCGGAGACGACA-5′ Commercially available purified human recombinant NRF2 (rNRF2) (Novus Biologicals, Littleton, CO, USA) was used as a positive control.

Techniques: Binding Assay, Control, Purification, Recombinant, Competitive Binding Assay, Mutagenesis, Super-Shift Assay

Journal: Antioxidants

Article Title: NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus

doi: 10.3390/antiox11081582

Figure Lengend Snippet: ChIP assay of endogenous CSE and NQO1 promoters. Chromatin from primary SAE cells infected with RSV for 20 h or treated with 25 µM tBHQ for 15 h was immunoprecipitated with anti-NRF2 antibody or IgG as negative control. qPCR was performed with immunoprecipitated DNA using primers spanning either the putative ARE site of the CSE promoter ( A ) or the NQO1 promoter ( B ). Fold change was calculated compared to IgG control. Data are expressed as mean ± SEM of three or four replicate data from two independent experiments (each circle represents individual data). * p < 0.05 relative to control cells.

Article Snippet: The sequences of oligonucleotides are shown: −156/−120 CSE wild type (WT) 5′-CGGTCGCGGTGACGTTTCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCCACTGCAAAGTCCGTTGCGGAGACGACA-5′ −156/−120 CSE mutant (M) 5′-CGGTCGCGG GT C AG T G TCAGGCAACGCCTCTGCTGT-3′ 3′-GCCAGCGCC CA G TC A C AGTCCGTTGCGGAGACGACA-5′ Commercially available purified human recombinant NRF2 (rNRF2) (Novus Biologicals, Littleton, CO, USA) was used as a positive control.

Techniques: Infection, Immunoprecipitation, Negative Control, Control