The second lysate was incubated with mock Dynabeads as a negative control. Then, the lysate was removed, and the beads were washed twice with lysis buffer, twice with 1 mL of wash
buffer (100 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA, and 0.5% sodium deoxycholate), and once with 1 mL of TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). The beads were incubated with 250 µL of TE plus 1% SDS for 10 min at 65 °C to elute DNA. The aspirated DNA solution was incubated at 65 °C overnight to reverse the cross-linking. The immunoprecipitated DNA and the input control DNA were treated with proteinase K, and precipitated with ethanol after proteins were removed with phenol–chloroform. Thus, purified DNA was dissolved in 30 µL of TE. The DNA sample (1 µL) was subsequently subjected to PCR in a total volume of 20 µL using gene-specific Nutlin-3a research buy primers (Supporting Information, Table S1). Preliminary reactions were performed to determine the optimal conditions to assure the linear amplification of each gene. In general,
PCR was carried out with 28 cycles of 94 °C for 15 s, 54 °C for 15 s, and 72 °C for 10 s with Ex Taq DNA polymerase (Takara Bio). PCR products (50~60 bp) were electrophoresed on an 8% polyacrylamide gel, stained with ethidium bromide, and photographed. The intensities of the bands in digitized images were quantified using the image j (1.42q) program, and the amounts of immunoprecipitated DNA were determined relative to the input DNA. Individual ChIP assays were repeated at least Buparlisib cost twice to confirm the reproducibility of the PCR-based experiment. Histidine-tagged Pdc2p(1–581) was expressed in bacterial cells and purified as described previously (Nosaka et al., 2005). The digoxigenin (DIG) gel shift kit (second generation; Roche Applied Science) was used for protein-DNA-binding assays. The oligonucleotide sequences used in this study are listed in Table S2. The
double-stranded DNA probe was prepared by heating at 95 °C for 5 min Demeclocycline and subsequent slow cooling to 65 °C. Then, the annealed fragment was isolated from a 5% polyacrylamide gel, and labeled by terminal transferase with DIG-11-ddUTP. The labeled probe (32 fmol) was incubated for 30 min at 25 °C with the recombinant Pdc2p(1–581) (2.5 µg) in 20 µL of EMSA buffer (20 mM HEPES pH 7.6, 30 mM KCl, 10 mM (NH4)2SO4, 1 mM EDTA, 1mM dithiothreitol, and 1% Tween 20) containing 1 µg of double-stranded poly(dI-dC), 1 µg of poly l-lysine, and 20 µg of BSA. The mixture was separated on an 8% polyacrylamide gel in 0.25× TBE and transferred to a nylon membrane (Biodyne B/Plus; Pall Gelman Laboratory) in 0.5× TBE using an electro-blotting system (Trans-blot SD Cell; Bio-Rad). Chemiluminescence of DIG-labeled DNA-protein complexes with anti-DIG-AP and CSPD on the nylon membranes was detected by an image analyzer (ImageQuant LAS 4000mini; GE Healthcare).