FIGURE 6.
GST pulldown assays of the interaction of Kap60 and Kap95 with Hxk2. The GST-Kap60 (a) and GST-Kap95 (b) fusion proteins were purified on glutathione-Sepharose columns. Equal amounts of GST-Kap60 and GST-Kap95 were incubated with cell extracts from wild-type, FMY304, and FMY305 strains. The yeast strains were grown in YEPD media until an A600 nm 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 Hxk2 variants were visualized on a Western blot with polyclonal anti-Hxk2 antibody (lanes 1–6). For the control samples, GST protein was also incubated with the high-glucose (H-Glc) cell extracts, but no signals were detected (lanes 7–9). The level of Hxk2 present in the different extracts used in a and b was determined by Western blot using anti-Hxk2 antibody. The Western blots shown are representative of results obtained from four independent experiments.