Association of Aft1 and Ssn6 with FRE1 and FRE2 promoters. (A) Wild-type and nhp6ΔΔ cells, carrying chromosomal AFT1-9Myc, grown in SCBPS, were subjected to ChIP with anti-Myc, followed by PCR analysis of the immunoprecipitated (IP) and input DNA using primers specific for FRE1, FRE2 and HTA1 promoters. (B) ssn6Δ* and ssn6Δ* aft1Δ cells, transformed with an HA-Ssn6 expressing plasmid, were grown and analyzed as above using anti-HA and primers specific for FRE1 and FRE2 promoters and ACT1 coding region. (C) ssn6Δ and ssn6Δ nhp6ΔΔ cells were transformed, grown and analyzed as above using anti-HA and primers specific for FRE1, FRE2 and TRP3 promoters. (D) ssn6Δ* cells transformed with an HA-Ssn6 expressing plasmid and grown under high-iron conditions (SC supplemented with 200 μM FeCl3 30 min prior to cell collection) were analyzed as above using anti-HA and primers specific for FRE1 and FRE2 promoters and ACT1 coding region. Bands in (A–D) were quantified using the PhosphorImager and ImageQuant software, and numbers express the indicated ratios. The specific recruitment of HA-Ssn6 on FRE1 and FRE2 promoters in ssn6Δ cells of two different genetic backgrounds was comparable.