rabbit anti cbd antibodies  (New England Biolabs)

Journal: PLoS ONE

Article Title: Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

doi: 10.1371/journal.pone.0008391

Figure Lengend Snippet: A , Titration of Keap1-mediated suppression of Nrf2 transcritional activity. NIH 3T3 cells were transfected with plasmids containing an ARE-dependent firefly luciferase reporter gene (ARE-Luc), expression plasmids for wild-type HA-Nrf2 and CBD-Keap1, and plasmids encoding Renilla luciferase to normalize transfection efficiency. The transfections were performed with 100 ng of ARE-Luc plasmid, 10 ng of pRL-TK, 40 ng of HA-Nrf2, and 1–40 ng of CBD-Keap1, as well as different amounts of pcDNA3.1 to maintain a total level of plasmid DNA at 200 ng. ARE reporter activity was measured 24 hours after the transfection. Results are representative of three independent experiments. * p <0.05 vs control pcDNA3.1. B , Effect of dh404 on Nrf2 nuclear translocation. NIH 3T3 cells were transfected for 24 hours with plasmids of HA-Nrf2 and CDB-Keap1 with a ratio of 10∶1 that resulted in complete suppression of Nrf2 transcriptional activity as indicated in A . The transfected cells were treated with dh404 (200 nM) in serum-free DMEM for 2 hours, and then subjected to immunofluorescence cofocal microscopic analysis. C , Cysteine residues in Keap1 required for dh404-mediated Nrf2 activation. NIH 3T3 cells were transfected for 24 hours with plasmids of HA-Nrf2 and CDB-Keap1 or plasmids of mutant Keap1 containing single cysteine-to-serine substitutions with a ratio of 10∶1 as indicated. The transfected cells were treated with or without dh404 (200 nM) in serum-free DMEM for 6 hours prior to ARE reporter assay. Results are representative of three independent experiments. * p <0.05 vs Nrf2 transfected group.

Article Snippet: Immunofluorescence staining was performed with rabbit anti-CBD antibodies (S6654S, New England Biolabs Inc.) and mouse anti-HA antibodies (F-7, sc-7392, Santa Cruz Biotechnology Inc.).

Techniques: Titration, Activity Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Translocation Assay, Immunofluorescence, Activation Assay, Mutagenesis, Reporter Assay

Journal: PLoS ONE

Article Title: Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

doi: 10.1371/journal.pone.0008391

Figure Lengend Snippet: A , HEK 293 cells that were transfected with plasmids of HA-Nrf2 (1000 ng), CBD-Keap1 (100 ng) and HA-Ub (1000 ng) for 24 hours were treated with or without dh404 (200 nM) in DMEM with 10% FBS for 2 hours as indicated. Ubiquitination of Nrf2 was determined by immunoprecipitation (IP) and immunoblot (IB) analysis using appropriate antibodies as indicated. Inputs of 10 µg of whole cell lysates were utilized to monitor the protein expression levels of target genes. B & C , HEK 293 cells that were transfected with plasmids of HA-Nrf2 (800 ng) and CBD-Keap1 (80 ng) for 24 hours were treated with or without dh404 (200 nM) and/or CHX (10 µg/ml) in DMEM with 10% FBS as indicated. D, HEK293 Cells were transfected with HA-Nrf2, CBD-Keap1 or mutant Keap1 as described above, and then stimulated with or without dh404 (200 nM) for 2 hours. Whole cell lysates were subjected to immunoblot analysis using anti(α)Nrf2, αKeap1 or αGapdah, as indicated. Results are representative of three independent experiments.

Article Snippet: Immunofluorescence staining was performed with rabbit anti-CBD antibodies (S6654S, New England Biolabs Inc.) and mouse anti-HA antibodies (F-7, sc-7392, Santa Cruz Biotechnology Inc.).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Mutagenesis

Journal: PLoS ONE

Article Title: Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

doi: 10.1371/journal.pone.0008391

Figure Lengend Snippet: HEK293 cells were transfected with plasmids of HA-Nrf2 (1000 ng), CBD-Keap1 (100 ng), HA-Cul3 (500 ng) or Myc-Rbx1 (400 ng) for 24 hours as indicated, and then were treated with or without dh404 (200 nM) in DMEM with 10% FBS for 2 hours as indicated. A , Recapitulation of Keap1/Cul3/Rbx1 complex-mediated Nrf2 degradation in HEK293 cells. B , Effect of dh404 on Keap1/Cul3/Rbx1 complex-mediated Nrf2 degradation in HEK293 cells. C , Effect of dh404 on assembling of Keap1/Cul3/Rbx1 complex in HEK293 cells. Cell lysates were subjected to immunoprecipitation (IP) and IB analysis using antibodies of Nrf2, Keap1, HA, Myc, and Gapdh as indicated. Results are representative of three independent experiments.

Article Snippet: Immunofluorescence staining was performed with rabbit anti-CBD antibodies (S6654S, New England Biolabs Inc.) and mouse anti-HA antibodies (F-7, sc-7392, Santa Cruz Biotechnology Inc.).

Techniques: Transfection, Immunoprecipitation

Journal: PLoS ONE

Article Title: Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

doi: 10.1371/journal.pone.0008391

Figure Lengend Snippet: HEK 293 cells were transfected with HA-Nrf2 and CBD-Keap1 as described in , and treated with dh404 (200 nM) in DMEM with 10% FBS as indicated. Immunoprecipitation (IP) and immunoblot (IB) analysis were performed using appropriate antibodies as indicated. Results are representative of three separated experiments. Cyo, cytosolic proteins; Nucle, nuclear proteins.

Article Snippet: Immunofluorescence staining was performed with rabbit anti-CBD antibodies (S6654S, New England Biolabs Inc.) and mouse anti-HA antibodies (F-7, sc-7392, Santa Cruz Biotechnology Inc.).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Antioxidants

Article Title: Therapeutic Administration of Oxcarbazepine Saves Cerebellar Purkinje Cells from Ischemia and Reperfusion Injury Induced by Cardiac Arrest through Attenuation of Oxidative Stress

doi: 10.3390/antiox11122450

Figure Lengend Snippet: ( A , C ) CBD-28k immunohistochemistry ( A ) and FJB histofluorescence ( C ) in the cerebellar vermis of the sham + vehicle ( A a, C a). The CA + vehicle ( A b–d, C b–d), sham + OXC ( A e, C e) and CA + OXC ( A f–h, C f–h) groups at 12 h and one and two days after CA/RoSC. In the CA + vehicle group, the numbers of CBD-28k-Purkinje cells are apparently reduced (arrows) in the Purkinje cell layer (PCL) two days after CA/RoSC, and FJB-cells are apparently increased (arrows) in the PCL at 12 h after CA/PCR. In the CA + OXC group, CBD-28k- and the number of FJB-Purkinje cells are markedly saved and reduced, respectively, two days after CA/RoSC. GCL, granular cell layer; MCL, molecular cell layer. Scale bar = 50 µm. ( B , D ). Numbers of CBD-28k-Purkinje cells ( B ) and FJB-Purkinje cells ( D ). The bars determine the means ± SEM ( n = 7, respectively; * p < 0.05 versus sham, † vehicle group; # p < 0.05 versus CA, † vehicle group).

Article Snippet: In short, the cerebellar sections were blocked with 20% normal goat serum (in 0.05 M PBS) and immunoreacted with primary rabbit anti-CBD-28k (diluted 1:1100; Cell Signaling Technology, Danvers, MA, USA), mouse anti-4HNE (diluted 1:450; Alexis Biochemicals, San Diego, CA, USA), sheep anti-SOD1 (1:1500; Calbiochem, Darmstadt, Germany) and sheep anti-SOD2 (1:1500; Calbiochem) at 4 °C for eight hours.

Techniques: Immunohistochemistry