Fig. 6. Fkh2p, Sep1p and Plo1p bind to PCB sequences in vivo. (A) Promoter regions amplified during the ChIP procedure together with the positions of the PCB and FLEX1 motifs. (B-E) Chromatin immunoprecipitation (ChIP) experiments with tagged versions of (B) Fkh2p-13myc, (C) Fkh2p-3HA, (D) Sep1p-13myc and (E) Plo1p-3HA, on the cdc15⁺, fkh2⁺ and plo1⁺ promoters. WCE, whole-cell extracts (non-immunoprecipitated input sample); IP, immunoprecipitates. Approximately ten times more of the precipitates were loaded than of the WCE input DNA. As negative controls, beads alone and normal mouse IgG were used for precipitations, and DNA of the act1⁺-coding sequence was used as substrate. For all Myc- and HA-tagged strains, control ChIPs were completed with a wild-type untagged strain. As a positive control for all four strains, histone H3 was analysed by ChIP with appropriate antibody on the cdc15⁺, fkh2⁺ and plo1⁺ promoters. In each case the ChIP was shown to be quantitative, because a serial reduction of the WCE input DNA resulted in a corresponding reduction of the observed PCR signal.