Figure 1.

The Repressor SEBF Interacts with the Transcriptional Activator Pti4.

(A) The yeast two-hybrid interaction between the SEBF bait and the Pti4 prey was revealed through the expression of the lacZ reporter gene (β-galactosidase activity), detected as a blue color (shown here in dark gray/black) on plates containing galactose (Gal) and supplemented with X-Gal (lane 1) or by prototrophic growth (activation of the LEU2 reporter gene) in medium lacking Leu but containing galactose (lane 3). The activation of the reporter genes was dependent on the expression of the prey construct, since its suppression in medium containing glucose (Glu) does not lead to the activation of the lacZ reporter gene (lane 2) or to prototrophic growth (lane 4).

(B) The pull-down assays were performed by incubating SEBF produced in E. coli and coupled to a solid support with Pti4 expressed as an HA fusion protein in yeast (lane 4). As negative controls, pull-downs were performed by omitting SEBF (lane 3) or Pti4 (lane 2). Lane 1 contains 100% of the amount of SEBF coupled to the solid support (bottom panel) or 20% of the amount of Pti4 used in the pull-down experiments (top panel). Proteins were analyzed by immunoblotting using an anti-HA antibody for Pti4 detection (top panel) or an anti-SEBF antibody (bottom panel).