HSP70 Transcription and Protein Aggregation Figure 1. HSF1 associates with p300 and p-TEFb after heat shock. (A and B) HeLa cells expressing pLKO.1 control (PKA scr) and PKAca shRNA (PKA kd) were incubated at 42uC then stained for p300, HSF1 and CDK9 using anti-p300, anti-HSF1 and anti-CDK9 antibodies respectively. Confocal microscopic analysis using secondary goat anti mouse alexa-488 (green), goat anti rat HSF1-Cy3 (red) and goat anti rabbit CDK9 (blue) was then performed. (C and D) Heat shocked cell expressing PKA scr (C) or PKA kd (D) were then stained for p300, HSF1 and RNA-phospho-Pol II S2 as above (E and F). Co-localization of HSF1, p300 and CDK9/RNA PolII S2 in NsB was quantitated by cell counting. Graphs represent mean +/2 SEM of 2 independent experiments. (G) Whole cell or nuclear lysates from control or heat shocked cells with or without an effective PKAca shRNA (PKA-kd-1) were immunoprecipitated with anti p300 antibodies and blotted for CDK9. The nitrocellulose membrane was stripped and then blotted with anti HSF1 antibodies. To control for off-target effects of PKAca shRNA, we used either a scrambled sequence (scr) or an shRNA determined to be ineffective PKA-kd-2. (H) HeLa cells were co-transfected with p300-HA and S320A/S320D-GFP constructs, p300 immunoprecipitated with anti-HA antibodies and HSF1 variants detected with anti-GFP Ab. Recombinant HSF1-GFP was used an electrophoretic mobility marker. Experiments were repeated reproducibly at least 3 times. doi:10.1371/journal.pone.0028950.g001 PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28950