FIGURE 2.

Identification of transcriptional activities of various human Fyn promoter regions and a ROS-responsive fragment in the Fyn promoter. A, the dual-luciferase (firefly and Renilla luciferase) expression plasmid pFynPromoterCheck vector map. The 4.6-kb XhoI/BglII and 1.2-kb XhoI/Hind fragments from the psiCHECK-2 plasmid (Promega) were ligated with the BglII/HindIII promoter fragment to construct the pFynPromoterCheck dual-luciferase expression plasmid. In these vectors, the Fyn promoter region DNAs act as the promoters for the Renilla luciferase gene. B, a schematic of Fyn promoter fragment positions, and relative transcriptional activities. The transcriptional activities of the promoter fragments listed were determined using luciferase assays of transfected 293T cells and normalized to the full-length Fyn promoter. C, identification of transcription activities using deletion analysis of the human Fyn promoter in 293T cells. The full length and a series of deleted Fyn promoter constructs were transfected into 293T cells for 24 h followed by dual-luciferase assays in which transcriptional activity of Fyn (Renilla luciferase activity) was normalized to firefly luciferase activity. The 0.4-kb SV40 early promoter was used as a positive control (all p values < 0.04). D, NAC increases Fyn promoter transcription activity in BaF3 cells. Diluent or 24 mm NAC was added to cell culture medium 2 h before the transfection of pFynPromoterCheck 0.2- and 0.1-kb vectors (all p values < 0.05). Luciferase assays were used to measure Fyn promoter activity. E and F, identification of a ROS-responsive fragment in the Fyn promoter. BaF3 p210 cells (F) or K562 cells (G) transfected with either -200 to -1 or -100 to -1 Fyn promoter fragments were treated with either diluent or 24 mm NAC followed by measurement of luciferase activity (all p values < 0.01 in E; all p values < 0.04 in G).