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

Western-blot analysis. Whole cell lysates were prepared by the addition of cold lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 2 mM EDTA) containing protease inhibitors (Nacalai Tesque). The protein concentration was determined using the Bio-Rad Protein Assay kit. Equal amounts of protein from each treatment were subjected to Western-blot analysis using specific antibodies against Oct-1 (Santa Cruz Biotechnology, sc-232) or b-actin (MBL, PM053). For PrP Sc detection, samples were digested with 40 mg/ml of proteinase K (PK; Sigma-Aldrich) at 37uC for 30 min and subjected to Western-blot analysis using anti-PrP antibody (Santa Cruz Biotechnology, M20). Chromatin immunoprecipitation (ChIP) assay. N2a58 cells grown in 100 mm dish were incubated with 1% formaldehyde for 10 min at 37uC to cross-link proteins to the DNA and washed with cold PBS buffer twice before lysis with SDS lysis buffer (50 mM Tris-HCl pH 7.5, 1% SDS, 10 mM EDTA) containing protease inhibitors. Cell lysates were sonicated on ice and the sheared chromatin was evenly divided and diluted in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris/HCl pH 8.1 and 150 mM NaCl) containing protease inhibitors. A small portion of each diluted chromatin was collected, protein-DNA cross-links were reversed, and samples were used as the input DNA control. The rest of chromatin suspension was prepared with Protein G-agarose slurry containing salmon sperm DNA (Millipore) and then incubated with either anti-Oct-1 or normal rabbit IgG antibodies (2 mg) overnight at 4uC with continual rotation. The Protein G-agarose slurry containing salmon sperm DNA was added to each chromatin solution and incubated for another 1 h at 4uC with constant rotation. The agarose beads were collected by centrifugation, washed and the antibody-bound chromatin was released from the agarose beads. Then the cross-link was reversed, samples were digested with PK and finally purified by phenol/chloroform extraction and ethanol precipitation. The DNA isolated from the immunoprecipitates was then subjected to PCR amplification. PCR was carried out as follows: 1 cycle at 94uC for 1 min; 25 cycles at 94uC for 30 s, 60uC for 5 s, 72uC for 30 s; and 1 cycle at 72uC for 7 min, using ChIP (2240/140) primers that amplify the region between nucleotides 2240 and 140 within the IRF-3 promoter. Then, nested PCR was carried out as follows: one cycle at 94uC for 1 min; 25 cycles at 94uC for 30 s, 60uC for 5 s, 72uC for 30 s; and one cycle at 72uC for 7 min, using ChIP (-119/-1) primers that amplify the region between nucleotides 2119 to 21 within the IRF-3 promoter. The amplified 119 bp DNA fragment was separated on 2% agarose gel and visualized with ethidium bromide.