FIG. 6.

Analysis of the interactions of Hap1 mutants with Hsp90. Extracts prepared from cells expressing wild-type or the indicated Hap1 mutant were incubated with streptavidin-conjugated magnetic beads (DYNAL) prebound with the biotinylated wild-type Hap1-binding site. The beads were extensively washed and boiled in SDS gel loading buffer to release the bound proteins (36). Hap1 and Hsp90 protein levels were detected by Western blotting. The membranes were first probed with an anti-Hap1 antibody and then stripped and probed with an anti-Hsp90 antibody. When a mutated Hap1-binding site was used in the pull-down assay, the amounts of bound Hap1 and Hsp90 proteins were undetectable or negligible (data not shown). These experiments were repeated at least twice.