Fig. 5. Sep1p binds to Fkh2p both in vivo and in vitro. (A) Fkh2p is co-immunoprecipitated with Sep1p from fission yeast extract. Sep1p-3HA was immunoprecipitated with antibody against HA from soluble extracts of fission yeast expressing Fkh2p-13myc from its endogenous promoter. The soluble extracts and the immunoprecipitates were analysed by western blotting with antibodies against HA and Myc (the latter to detect Fkh2p-13myc). As controls, extracts from Sep1p-3HA-expressing cells alone and precipitates without antibodies (beads only) from the double-tagged strain are also included. Approximately ten times more of the precipitates were loaded than of the soluble input fractions. Arrow indicates Fkh2p-13myc co-immunoprecipitated with Sep1p-3HA. (B) 6His-Fkh2p is pulled down by GST-Sep1p in vitro. Bacterially expressed 6His-Fkh2p and GST-Sep1p were purified and mixed together. When GST-Sep1p was bound to GST beads, 6His-Fkh2p was present in the pull-down fraction, whereas GST alone was unable to pull down 6His-Fkh2p. Arrow indicates pulled-down 6His-Fkh2p with GST-Sep1p.