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2016-03-05 发布于广东
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a transgenic mouse model for monitoring oxidative stress.doc

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a transgenic mouse model for monitoring oxidative stress

A transgenic mouse model for monitoring oxidative stress. Daisuke Oikawa1, 2, 3, Ryoko Akai1, 2, Mio Tokuda2, Takao Iwawaki1, 2, 4 * 1 Iwawaki Laboratory, Advanced Scientific Research Leaders Development Unit, Gunma University 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan 2 Iwawaki Initiative Research Unit, Advanced Science Institute, RIKEN 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 3 Research Fellow of the Japan Society for the Promotion of Science 8 Ichiban-cho, Chiyoda-ku, Tokyo 102-8472, Japan 4 PRESTO, Japan Science and Technology Agency 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan * Correspondence and requests for materials should be addressed to Takao Iwawaki (iwawaki@gunma-u.ac.jp) Supplementary Figures Supplementary Figure 1 Optimisation of the luciferase-fused Nrf2 fragment. (a) Schematic of trial constructs. A partial fragment (a.a.1-93 or a.a.1-433) or full length of human Nrf2 (purple) was fused to Flag-tagged luciferase (GL4; yellow). These fusion genes were driven by 3xARE promoter (light blue), or HSV-TK (Herpes simplex virus thymidine kinase) promoter (grey). (b) Luciferase assay with trial constructs. Each trial construct was transfected into HeLa cells, and luciferase assays were performed after treatment with or without various stresses (sodium arsenite (ASN), diethylmaleate (DEM), H2O2) for 8 hr or 16 hr. Supplementary Figure 2 Effect of OKD48 construct on endogenous Nrf2-Keap1 pathway in vitro. (a) Protein expression of the OKD48 construct under oxidative stress. The OKD48 construct was transfected into HEK293T cells, and then treated with or without sodium arsenite (ASN), diethylmaleate (DEM), or H2O2 for 8 hr. Their lysates were subjected to anti-Luc, anti-Keap1 or anti-GAPDH Western blotting. (b) Induction of HO-1 mRNA in OKD48-transfected cells. The OKD48 construct was transfected into HeLa cells, and then treated with or without sodium arsenite (ASN), diethylmaleate (DEM), or H2O2 for 8 hr. Their total RNAs were subjected to