Figure 5. Reduced HSF1 levels in cells expressing polyQ-expanded Htt.

A) Western blots prepared with protein lysates from STHdh(Q7) and STHdh(Q111) cells that were grown at 33°C or heat shocked (HS, 42°C for three hours). The blots were probed with anti-HSF1 antibodies and anti-tubulin antibodies as a loading control. B) Upper panel: quantification of Western blots as shown in Figure 5 A. Signals of total HSF1 were normalized to the corresponding tubulin signal in each experiment. Lower panel: quantification of Western blots as shown in Figure 5 A. Signals of the top, i.e. the heat shock-induced part of HSF1 were normalized to the corresponding tubulin signal in each experiment. The signal of the top part of HSF1 at 33°C was set as 1 for each condition. Error bars represent SDs. Results from three independent experiments were analyzed. *p<0.07 (two-tailed t-test). C) Cross-linking of HSF1. Protein lysates were prepared as in A) followed by cross-linking with EGS (see Materials and Methods for details). The ensuing Western blots were probed with and anti-HSF1 antibody. D) Quantification of three independent experiments as shown in C). The error bars represent SDs. * p<0.01 (two-tailed t-test). E) Protein lysates were prepared as in A) and then separated into cytosolic and nuclear fractions. The blots were probed with anti-HSF1 antibodies and anti-lamin A/C antibodies and tubulin antibody as a loading control and as a control for the purity of the fractions (nucleus and cytosol respectively). F) Viability assay (luciferase activity, Promega) of STHdh(Q7) and STHdh(Q111) cells that were grown at 33°C and heat shocked cells (three hours at 42°C) in the absence or presence of triptolide. The signal from STHdh(Q7) cells grown at 33°C without triptolide was set as 100% and the other signals were calculated accordingly. The error bars represent SDs. Results from four independent experiments were analyzed. * p<0.001 (two-tailed t-test).