Tec1 forms a complex with Ste12 and Dig1 but not with Dig2. (A) Immunoprecipitation of Tec1-HA. Protein lysates were subjected to immunoprecipitation with an anti-HA antibody, and the precipitation products were resolved by SDS-polyacrylamide gel electrophoresis and probed with an anti-myc antibody. As controls, cell lysates were subjected to Western blotting with anti-myc and anti-HA antibodies. The following yeast strains were used: lane 1, HLY3334 (TEC1-HA STE12-myc); lane 2, HLY3335 (TEC1-HA DIG1-myc); lane 3, HLY3336 (TEC1-HA DIG2-myc); lane 4, HLY3320 (TEC1-myc); lane 5, HLY3321 (STE12-myc); lane 6, HLY3322 (DIG1-myc); and lane 7, HLY3323 (DIG2-myc). The lower band in lane 2 is a breakdown product of Dig1-myc. (B) Immunoprecipitation of Dig2-HA, as in panel A. The yeast strains in each lane are as follows: lane 1, HLY3337 (DIG2-HA STE12-myc); lane 2, HLY3338 (DIG2-HA DIG1-myc); lane 3, HLY3339 (DIG2-HA TEC1-myc); lane 4, HLY3327 (DIG2-HA); lane 5, HLY3321 (STE12-myc); lane 6, HLY3322 (DIG1-myc); and lane 7, HLY3324 (TEC1-HA). The lower bands in lanes 2 and 6 are a breakdown product of Dig1-myc. (C) Tec1 binds to the N-terminal region of Ste12. The yeast strain HLY3350 (ste12 tec1) carrying plasmid pHL754 (ADH1p-Ste12(1-215)-myc) was transformed with either pHL731 (ADH1p-TEC1-HA) or a vector (pRS313), and the transformed strains were used in immunoprecipitation with an anti-HA antibody. The precipitated proteins were analyzed by Western blotting with an anti-myc antibody. IB, immunoblot.