Fig. 3.

Mutation of HSF1 K80 inhibits the HSR. (A) Mutation of HSF1 at K80 disrupts DNA binding activity. EMSA reactions were performed with extracts from hsf1^−/− cells transfected with the indicated HSF1 constructs treated with or without heat shock (HS) (top). The EMSA probe contains the proximal HSE from the human hsp70 promoter. Western blot analysis was performed on the same samples to show HSF1 and Hsc70 levels. (B) Mutation of recombinant HSF1 at K80 disrupts DNA binding ability. EMSA reactions with increasing amounts (5, 20, 40, 80, or 120 ng) of recombinant WT HSF1 or HSF1 K80Q and a probe containing an HSE are shown (top). A sample without (−) HSF1 protein was a control. Western blot analysis was performed on the same samples to show HSF1 expression levels. (C) Failure of HSF1 mutated at K80 to rescue the HSR in hsf1^−/− cells. hsf1^−/− cells were transfected with the indicated versions of human HSF1 and treated with or without heat shock. RNA was quantified using qPCR with primers for the indicated genes. Data are normalized to values obtained for glyceraldehyde 3-phosphate dehydrogenase and are relative to the abundance of each mRNA in WT HSF1 cells treated without heat shock (value set as 1). Experiments in (A) to (C) were performed in triplicate, and error bars indicate ±SD.