Figure 3

Comparative analyses in HSF1-negative mouse embryo fibroblasts. (A) Comparison of DNA-binding abilities of HSF1 and HSF1 mutant S326A. An electrophoretic mobility shift assay was carried out using an HSE DNA probe and extracts from heat-treated or not-heat-treated cells that had been transfected one day earlier with the constructs indicated on top of the gel. An anti-HSF1 western blot of the same samples reporting on the relative levels of expression of HSF1 forms from the different constructs is shown below the gel. HSF1/HSE: HSF1-DNA complex; NS: nonspecific signal, serving as a loading control for the group of samples from not-heated cells, and, independently, for the group of samples from heat-treated cells; HS: heat-treated for 30 min at 43°C; C: not heat-treated. (B) Heat-induced transactivation of endogenous hsp70 gene(s) in cells transfected with small amounts of the constructs indicated above the blots and with luciferase reporters HSP70-fLUC and pRL-CMV. One day after transfection, cultures were either left untreated (C) or were heat-treated for 30 min at 43°C (HS). Extracts were prepared after 6 h of further incubation at 37°C and were used for western blots. The top blot was probed with an antibody recognizing HSP70, whereas the bottom blot was probed with HSF1 antibody. The bottom blot demonstrates that mutant and wildtype HSF1 forms accumulated to similar levels. Note the presence of a weak nonspecific signal present in all lanes that happened to co-migrate with the transfected HSF1 forms. (C) Quantitative comparisons of data of B. (D) Relative luciferase reporter activities in the same extracts that were analyzed for HSP70 in B. Representative results from one of several independent experiments are shown in this Figure.