Figure 5. CE2 is sufficient to bind Hsp70.

(A) Fluorescence polarization assay of CE2 and ce2-mut peptides labeled with 5-carboxyfluorescein (5-FAM) and recombinant Hsp70 (Ssa2). Peptides were maintained at 100 nM while Hsp70 was titrated at the indicated concentrations. Reactions were incubated for 30 min at room temperature prior to measurement. (B) Schematic of the ‘decoy’ assay. Overexpression of mKate-based decoy constructs activates Hsf1 in trans by titrating away Hsp70. (C) Decoy assay results. Cells bearing the indicated decoy constructs were induced with 1 µM estradiol for 16 hr at 30°C and measured by flow cytometry. The HSE-YFP values in each cell were normalized by the expression of the decoy as measured by mKate fluorescence. Data are represented as median values of 10,000 cells relative to the median value of mKate alone. Error bars are the standard deviation of 3 biological replicates. Statistical significance was determined by one-way ANOVA (*p<0.05; **p<0.01).