HSF1 and Protein Kinase A Figure 5. PKAca knockdown inhibits HSF1 association with the hsp70.1 promoter. (A) Diagram of primers mapping to the hsp70.1 gene. B) ChIP experiments. HeLa cells either with PKA knockdown (PKA kd #1) or expressing a scrambled control RNA (PKA scr) were incubated without or with heat shock (43uC, 1 hr). Cells were then fixed with formaldehyde, sonicated and analyzed by ChIP assay using HSF1 antibodies or preimmune- IgG as control. The immunoprecipitated DNA was PCR amplified using the primers specific to the promoter (HSE, upper lane) or ORF (EXN, bottom lane) sequences in hsp70.1. Each panel (from left to right) shows the PCR products from the HSF1 ChIP assay (anti-HSF1), IgG ChIP or from control sample of genomic DNA (input). Experiments were performed three times with similar findings. C) ChIP PCR products were also analyzed using the ABI 7300 real time PCR system and the fold increase by 22DDCT method. Levels of HSF1 ChIP PCR products were compared with the products associated with pre-immune-IgG and plotted for the respective regions of hsp70.1. ChIP experiments were each performed at least 4 times with and data plotted as mean + SD with consistent findings. Differences in gene occupation by HSF under the different conditions were analyzed statistically by Student’s t test. Promoter occupancy by HSF1 after heat shock was significantly higher than in PKAca knockdown cells or occupancy of Hsp70EXN regions with or without PKAca and significance is indicated: *** p,0.001 (left panel). Differences in HSF1 binding to promoter (HSP70HSE) or ORF regions (HSP70EXN) in non-heat shocked cells were not significant (p.0.05). D) Luciferase assay of Hsp70.1 promoter-reporter activation in response PLoS ONE | www.plosone.org 10 November 2010 | Volume 5 | Issue 11 | e13830