Persistent prion infection disturbs the function of Oct-1, resulting in the down-regulation of murine interferon regulatory factor-3

Homma, Takujiro; Ishibashi, Daisuke; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Satoh, Katsuya; Atarashi, Ryuichiro; Nishida, Noriyuki

Transient transfection and reporter assay. Cells were seeded at densities of 2 3 10 5 cells per well in 24-well plates and grown in a humidified incubator at 37uC and 5% CO 2 overnight to 70-80% confluence. In basal promoter activity studies, cells were co-transfected with 500 ng of reporter plasmids and 100 ng of renilla luciferase control plasmid (pRL-null Vector; Promega). For ectopic expression, cells were additionally co-transfected with 500 ng of pcDNA Oct-1-HA or pcDNA (empty plasmid). Transfections were performed using Lipofectamine LTX (Invitrogen) following the manufacturer's instructions. After 24 h, cells were lysed using Passive Lysis Buffer (Promega) and the luciferase activity was quantified by the Dual Reporter Assay System (Promega) and Mithras LB940 luminometer (Berthold Technologies) according to the manufacturer's instructions. Results were normalized to the coexpressed renilla luciferase activity.