Fig. 2. Plo1p binds to Mbx1p in vivo. (A) Two-hybrid interaction between Plo1p and Mbx1p. Wild-type plo1⁺ in the two-hybrid bait vector was transformed into budding yeast, together with empty prey vector, or prey vector containing either mbx1⁺, fkh2⁺, sep1⁺ or sck1⁺ (the latter encoding a known Plo1p-interacting protein) (Reynolds and Ohkura, 2003). Colour reactions were quantified in cells grown in liquid medium and are plotted; error bars indicate two standard errors. Table indicates combinations of two-hybrid plasmids used. (B) Two-hybrid interaction between Plo1p and Mbx1p requires both the kinase and the Polo-box domains. Wild-type plo1⁺ or either kinase (plo1K69R) or Polo-box domain (plo1.472-684 and plo1DHK625AAA) mutants of plo1 in the two-hybrid bait vector were transformed into budding yeast, together with the empty prey vector, or the prey vector containing either mbx1⁺ or sck1⁺. Colour reactions were quantified in cells grown in liquid medium and are plotted; error bars indicate two standard errors. Table indicates combinations of two-hybrid plasmids used. (C) Mbx1p is co-immunoprecipitated with Plo1p from fission yeast extract. Plo1p was immunoprecipitated with antibody against the native protein from soluble extracts of fission yeast expressing Mbx1p-13myc from its endogenous promoter. The soluble extracts and the immunoprecipitates were analysed by western blotting with antibodies against Plo1p and Myc (the latter to detect Mbx1p-13myc). As controls, extracts from mbx1 and precipitates without antibodies (beads only) from tagged strains are also included. Approximately ten times more of the precipitates were loaded than of the soluble input fractions. Arrow indicates co-immunoprecipitated Mbx1p-13myc with Plo1p.