1 hour) and fixed cells were later stained for PKAca and HSF1 with anti- PKAca and anti-HSF1 antibodies as described in A. E) HeLa (PKAkd#1) cells heat shocked (43oC, 1 hour) or not. Cells were then stained for PKAca and HSF1 using the antibody cascades employed in A–D. All experiments were repeated at least once with consistent findings. Found at: doi:10.1371/journal.pone.0013830.s002 (4.76 MB TIF) Figure S3 Quantitative analysis of the distribution of HSF1 in wt HeLa, HeLa (PKA scr) and HeLa (PKA kd#1) cells under non- treated, heat shock or LMB treated conditions as indicated in figure was performed after confocal fluorescence microscopy. The sub-cellular distribution of HSF1 was scored according to whether the protein was detected in the nucleus, in both nucleus and cytoplasm or strictly in the cytoplasm. Found at: doi:10.1371/journal.pone.0013830.s003 (0.13 MB TIF) Figure S4 PKAca associates with pS320 HSF1 in different activation conditions. 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