Fig. 7. Requirement of Plo1p, Mbx1p and Sep1p for Fkh2p promoter binding in vivo. ChIP experiments with Fkh2p-13myc and Fkh2p-3HA from extracts of cells containing mutants of plo1, mbx1 and sep1. (A-C) Crosslinked DNA was prepared from (1) wild-type and (A) plo1-ts35 cells at (2) permissive and (3) restrictive temperatures, or from cells with chromosome deletions of (B) mbx1 or (C) sep1. In each case, Fkh2p-13myc and Fkh2p-3HA were analysed by ChIP using anti-Myc and anti-HA antibodies respectively, and binding to PCB promoter fragments from cdc15⁺, fkh2⁺ and plo1⁺ detected by PCR. 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. As a positive control, histone H3 was analysed by ChIP with appropriate antibody. Graph in A shows the quantification of Fkh2p binding against WCE input DNA. 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 (data not shown).