HSF1 and Protein Kinase A Figure 1. HSF1 binds to PKAca in cells. A) Immunoprecipitation assays. HeLa cells were incubated for 1 hour at 37uC or 43uC before they were harvested on ice and equalized for protein concentration. Cell extracts were then probed by immunoprecipitation with either anti-PKAca (lane 1,2) anti-PKARI (lane 3), anti-PKARII antibodies (lane 4) or rabbit IgG. Immunoprecipitates were then analyzed by 10% SDS-PAGE and immunoblot with anti-HSF1 antibodies. We have shown the IgG heavy chain to indicate the equal loading of immunoprecipitates in this experiment. The membrane was then stripped and re-probed sequentially with anti- PKAca, PKARI and PKARII antibodies. We also analyzed the levels of HSF1, PKAca, PKARI and PKARII in the cell lysate (prior to immunoprecipitation) and these data are shown beneath the immunoprecipitation data. Experiments were carried out in duplicate with similar findings. B) Immunoprecipitation assay with anti-HSF1 antibodies in MCF-7 cells. MCF-7 cells were incubated for 1 hour at 37uC or 43uC. Other cultures were simultaneously treated with growth factors (30 mM HRG or 100 ng/ml IGF-1 for 24 hr) or inhibitors (2 mM 17AAG for 24 hr, 5 mM MG-132 for 6 hr) as indicated on the figure (panels 3–6). Cell lysates were then prepared as in (A). Immunoprecipitation was performed with anti-HSF1 antibodies and immunoprecipitates later analyzed by immunoblot with anti- PKAca antibodies. Equal amounts of immunoprecipitated proteins were loaded and immunoblotted with anti-HSF1 and anti- PKAca antibodies. Again, we have shown the IgG heavy PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e13830