Questions 1 What molecules are present in the regions of the gel corresponding to bands A, B, and C? 2. What would the autoradiogram look like if the samples had been electrophoresed in an SDS- polyacrylamide gel? 3. Describe the effect of heat shock on the cells (compare samples 1 and 2). 4. Describe the effect of indomethacin on the cells (compare samples 1-6). 5. What was the aim of using the antibodies? What is the role of HSF-1 and HSF-2 in the effect of indomethacin? 6. We cannot determine from this experiment whether indomethacin affects the expression of HSFs or the activity of HSFs. What technique could be used to identify whether HSF expression or HSF activity is affected by indomethacin? Indomethacin (μM) α-HSF1α-HSF2 C HS 100 250 500 1000 500 1000 500 1000 + В CB A 1 2 3 4 5 6 7 8 9 10 Samples

Experiment: 

Heat shock factors (HSF) are transcription factors that induce genes encoding proteins that protect against cellular stress. Two distinct heat shock factors, called HSF1 and HSF2, recognize the same binding site in DNA. The figure shows the result of an experiment in which the effect of an anti-inflammatory drug, indomethacin, was studied. HeLa cells were cultured at 37°C (lane 1 [C, control] and lanes 3-10), or 42° C (lane 2; HS, heat-shocked). The cultures for samples 3- 10 were treated with various concentrations of indomethacin, indicated at the top of each lane. After treatment, nuclear protein extracts were prepared and mixed with 32P-labeled, double- stranded oligonucleotides containing the HSF binding site. Anti-HSF1 (samples 7 and 8) or anti- HSF2 (samples 9 and 10) antibodies were also added to some of the samples. The samples were subjected to electrophoresis in a nondenaturing polyacrylamide gel, and autoradiography was performed.