Figure 3.

Hsp90 and Tah1 stabilize Pih1 in vivo. (A) The R2TP complex was isolated from log- and stationary-phase WT and tah1Δ cells expressing a chromosomal copy of PIH1–3FLAG using αFLAG resin. The interaction of Hsp90 with the complex was assessed by Western blot analysis. (B) The change in the levels of Pih1, Rvb2, Rvb1, Hsp90, and Tah1 in different strain backgrounds was monitored by Western blot analysis at three growth stages as indicated by OD₆₀₀. Cells had a chromosomal copy of PIH1–3FLAG in a WT background (sample 1; PIH1–3FLAG), in a background deleted of TAH1 containing an empty vector (sample 2; PIH1–3FLAG, tah1Δ, and p414), or in a background deleted of TAH1 but expressing Tah1 from a p414 plasmid (sample 3; PIH1-3FLAG, tah1Δ, and p414Tah1). 6.5 μg of total proteins was typically loaded and blotted with specific antibodies. (C) WT or deg-TAH1 yeast cells were exposed to heat shock for 2 h in the presence of 40 μg/ml cycloheximide. For each sample, proteins from cells with equivalent OD₆₀₀ were loaded onto SDS-PAGE gels and blotted with specific antibodies.