Fig. 8. Nucleolar localization of Hsp70 correlates with the activation and deactivation of HSF1. (A) An autoradiograph image of proteins interacting with Myc-HSF1 in labeled 4H9 cells. Anti-Myc antibodies were used for immunoprecipitation. K562 cells were used as a negative control. The two obtained bands were detected also by silver staining and analyzed by matrix-assisted laser desorption ionization mass spectrometry. (B) Coimmunoprecipitation of endogenous HSF2 and Hsp70 with endogenous HSF1 in heat-shocked HeLa cells. Monoclonal HSF1 antibodies were used for immunoprecipitations (IP). Cells were untreated (C) or heat shocked for 30 minutes to 6 hours (30'-6h). Thermotolerance was obtained by a 1-hour heat shock, followed by 1 or 3 hours of recovery (HS+1R or HS+3R). Thermotolerant cells were heat shocked for 30 minutes or 1 hour (HS+3R+HS30' or HS+3R+HS1h). The bottom blots (WB) show the expression levels of HSF1, HSF2 and Hsp70. Hsc70 was a control for equal loading. Arrows indicate the inducibly phosphorylated HSF1. (C) Fluorescence micrographs showing the localization of HSF1 and Hsp70 in untreated (C), heat-shocked (HS) for 1 hour or 6 hours, thermotolerant (HS+3R), and heat-shocked thermotolerant HeLa cells (HS+3R+HS1h). Merge lane indicates possible co-localization (yellow) and DNA is stained by DAPI (blue). Scale bar, 5 µm.