Figure 5

Chromatin immunoprecipitation assays. A-Formaldehyde cross-linked WCEs were immunoprecipitated with anti-14-3-3ε antibody. Approximately the same amount of wild type and mutant 14-3-3ε were revealed by the anti-14-3-3ε antibody. NRS and 14-3-3ε WCE were used as controls for the IP. B- The immunoprecipitated chromatins were subjected to conventional PCR and resolved in a 1.5% agarose gel. Upper panel of the gel shows the PCR products of the ChIP performed with wild-type 14-3-3ε extracts (arrow). The lower panel shows the PCR products of the ChIP performed with α5-helix-deleted 14-3-3ε extracts (arrow). Template DNAs from reverse cross-linked cells immunoprecipitated with anti-14-3-3ε antibody (lane 1) or pre-immune sera (lane 2), wild-type and mutant yeast WCE (lane 3 in upper and lower panel respectively); 10 ng of genomic DNA (lane 4) and 10 ng of plasmid pRS306 containing ARS307 (line 5), were used as controls. C- The amounts of wild-type or α5-deleted 14-3-3ε proteins associated with ARS307, ARS1 and their negative regions, Neg307 and R2.5 respectively, were measured by Real-time PCR. Cross-linked WCE were immunoprecipitated using anti-14-3-3ε antibody or NRS, and each corresponding ChIP material was used as template DNA. Error bars represent the average of three separate experiments and one standard deviation.