Journal: Cell death and differentiation

Article Title: Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis.

doi: 10.1038/sj.cdd.4401025

Figure Lengend Snippet: Figure 3 Ref-1 inhibits H/R-induced, and rac1-regulated oxidative stress. (A) Intracellular H2O2 levels in uninfected, Adbgal-, and Adref-1-infected cells, under normoxia, and with H/R and TNF. *P50.05 compared with Adbgal. Reoxygenation was for 5 min, and TNF 15 min. (B) Intracellular H2O2 levels 5 min after addition of exogenous H2O2 in Adbgal-, and Adref-1-infected cells. (C) Intracellular H2O2 levels in cells co-infected with Adbgal-, Adrac1V12-, and Adref-1. Inset shows adenoviral-induced co-overexpression of ref-1 and rac1V12. The figures are representative of two separate experiments

Article Snippet: Nuclear and cytoplasmic protein was extracted and fractionated as previously described.49 Western blots were performed with an antibody against ref-1 (SC-334, Santa Cruz Biotechnology), or rac1 (23A8, UBI) and bound immunocomplexes were visualized by enhanced chemiluminescence (Amersham).

Techniques: Infection, Over Expression

Journal: Cell death and differentiation

Article Title: Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis.

doi: 10.1038/sj.cdd.4401025

Figure Lengend Snippet: Figure 4 Ref-1 inhibits H/R-induced, rac1-regulated NF-kB activity by inhibiting its nuclear translocation. (A) NF-kB transcriptional activity measured by reporter assay in uninfected, Adbgal-, and Adref-1-infected cells. Values are expressed as fold induction compared with Adbgal-infected cells. *P50.05 compared with Adbgal. The data is representative of three separate experiments. (B) EMSA for NF-kB DNA binding in Adbgal-, and Adref-1-infected cells. Where indicated, 1006 excess unlabelled kB oligonucleotide (comp kB), mutant kB oligonucleotide (kB (M)), or antibody to the p50 subunit of NF-kB (ap50) were added to the binding mixture. The figure is representative of two separate experiments. (C) NF-kB reporter assay in cells co-infected with Adbgal-, Adrac1V12-, and Adref-1. *P50.05 compared with AdRac1v12/Adbgal. Immunostaining for the p50 (D) and p65 (E) subunits of NF-kB in Adbgal-, and Adref-1-infected cells under normoxia (D,E), after H/R (D,E), and after exogenous H2O2 addition (E). Reoxgenation and H2O2 treatment were for 1 h. Arrows indicate nuclei. The figures are representative of two separate experiments

Article Snippet: Nuclear and cytoplasmic protein was extracted and fractionated as previously described.49 Western blots were performed with an antibody against ref-1 (SC-334, Santa Cruz Biotechnology), or rac1 (23A8, UBI) and bound immunocomplexes were visualized by enhanced chemiluminescence (Amersham).

Techniques: Activity Assay, Translocation Assay, Reporter Assay, Infection, Binding Assay, Mutagenesis, Immunostaining

Journal: Cell death and differentiation

Article Title: Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis.

doi: 10.1038/sj.cdd.4401025

Figure Lengend Snippet: Figure 5 Nuclear and Cytoplasmic Roles of ref-1. Summary diagram illustrating the cytolasmic and nuclear effects of ref-1. *Signifies the role of ref-1 in suppressing rac1-regulated ROS production demonstrated in this study. Figure does not include other potential mechanisms by which ref-1 may suppress NF-kB activation, nor does it include other potential extra-nuclear actions of ref-1. AP: Apurinic/Apyrimidinic

Article Snippet: Nuclear and cytoplasmic protein was extracted and fractionated as previously described.49 Western blots were performed with an antibody against ref-1 (SC-334, Santa Cruz Biotechnology), or rac1 (23A8, UBI) and bound immunocomplexes were visualized by enhanced chemiluminescence (Amersham).

Techniques: Activation Assay