Flo8 and Mss11 act on UAS2-1 by interacting with Ste12 and Tec1. (A) The four activators interact with each other. Cells carrying plasmids which express LexA fusion proteins or HA-tagged proteins were grown in synthetic medium containing 2% glucose. Protein extracts (500 μg) were immunoprecipitated with anti-HA antibody. The precipitates and input extracts (50 μg) were separated by SDS-8 to 10% PAGE and analyzed by Western blot analysis with LexA antibody. The membranes were stripped and reprobed with anti-HA antibody. +, present; −, absent. (B and C) Flo8 and Mss11 binding to UAS2-1 requires Ste12 and Tec1. FLO8-HA wild-type (WT), FLO8-HA ste12Δ, MSS11-HA wild-type, MSS11-HA ste12Δ, or untagged strains bearing pLG-UAS2-1 were grown in synthetic medium containing 2% glycerol-ethanol to mid-log phase and subjected to anti-HA (α-HA) ChIP assays as described for Fig. 2B.