Fig. 8. Cell-cycle-specific binding of Fkh2p, Sep1p and Plo1p to PCB promoter DNAs. (A-C) Separate populations of (A) cdc25-22 fkh2-3HA, (B) cdc25-22 sep1-13myc and (C) cdc25-22 nmt41:plo1-3HA cells were synchronised by transient temperature arrest, and samples taken every 20 minutes after returning the cells to the permissive temperature for ChIP and northern blot analysis. Septation indices were counted microscopically and are plotted to indicate the synchrony of each culture. Crosslinked DNA was prepared from each sample and Fkh2p-3HA, Sep1p-13myc and Plo1p-3HA were analysed by ChIP using anti-HA and anti-Myc antibodies. Binding of Fkh2p-3HA, Sep1p-13myc and Plo1p-3HA to the cdc15⁺, fkh2⁺ and plo1⁺ promoters was detected by PCR. As a loading control, PCR was performed with 10% whole-cell extracts (WCE; non-immunoprecipitated sample) containing input DNA, and plo1⁺ (A, B) and cdc15⁺ (C) oligonucleotides. Graphs show the quantification of Fkh2p, Sep1p and Plo1p binding against WCE input DNA. RNA was prepared from duplicate samples to detect mRNA levels of cdc15⁺, fkh2⁺ and plo1⁺ by northern blot analysis. mRNA levels of cdc22⁺, a known G1-S phase expressed transcript independent of PBF-PCB controls were also detected to allow comparison between experiments. Quantification of each transcript against rRNA is shown under each northern blot panel. asy, control DNA and RNA samples from asynchronous cells prior to synchronisation.