Interaction of Kap60 and Kap95 with Mig2. In vivo co-immunoprecipitation of Kap60 and Kap95 with Mig2. The wild-type, FMY501 (Mig2-GFP) (a) or a control strain, (TetR-GFP) synthetizing GFP only (b) were grown in YEPD-media until an A600nm of 0.8 was reached and then shifted to low glucose (L-Glc) conditions for 1 h. The cell extracts were immunoprecipitated with a polyclonal anti-Kap60 or anti-Kap95 antibody (lanes 3-6) or a polyclonal antibody against Pho4 (lanes 1 and 2). Immunoprecipitates were separated by 12% SDS-PAGE, and co-precipitated Mig2-GFP was visualized by Western blot with polyclonal anti-GFP antibodies. The level of immunoprecipitated Kap60 or Kap95 in the blotted samples was determined by using anti-Kap60 and anti-Kap95 antibodies, respectively. The level of Mig2-GFP and GFP present in the different extracts was determined by Western blot using anti-GFP antibody. The Western blots shown are representative of results obtained from four independent experiments. The GST-Mig2 fusion protein was purified on glutathione-Sepharose columns. Equal amounts of GST-Mig2 were incubated with cell extracts from the wild-type strain W303-1A. The yeasts were grown in YEPD media until an A600nm of 0.8 was reached and then shifted to low (L-Glc) glucose conditions for 1 h. After exhaustive washing the proteins were separated by 12% SDS-PAGE, and retained Kap60 and Kap95 proteins were visualized by Western blot using polyclonal anti-Kap60 (c) and anti-Kap95 (d) antibodies respectively. For the control samples, GST protein was also incubated with high- (H-Glc) and low-glucose (L-Glc) cell extracts, but no signals were detected. The level of Kap60 and Kap95 proteins present in the different extracts used in Figure 4c and4d was determined by Western blot using anti-Kap60 and anti-Kap95 antibodies respectively. The Western blots shown are representative of results obtained from four independent experiments.