Analysis of E2F1 binding on the RIP140 promoter.

<p>(<b>A</b>) Electromobility shift assay was used to analyze E2F1/DP1 binding on the adenoviral E2F response element (Ad2E2F) or on the E2Fa, b, c, d and e sites of the RIP140 promoter (viewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035839#pone-0035839-g001" target="_blank">Figure 1</a>). Asterisks indicate the retarded bands which contain E2F1. (<b>B</b>) Dose response experiment using increasing amounts of E2F1/DP1 on E2Fa, d and e binding sites. (<b>C</b>) ChIP experiments using immunoprecipitation (IP) of E2F1 on the E2Fa and E2Fd sites of the human RIP140 promoter. The cyclin A2 promoter was used as a positive control. Data are expressed as percent of the PCR signal obtained with the amount of chromatin used for IP (input). Negative control of IP was performed without any antibody which, in our hands, gave the same background as an isotype-matched mAb. Statistical analysis was performed using the Student t test (*p<0.05 and **p<0.005). (<b>D</b>) Human RIP900-luc (25 ng), E2F1 to 6 (25 ng) and DP1 (25 ng) were overexpressed in the indicated combinations. Relative luciferase activity was normalized with renilla luciferase activity as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035839#s2" target="_blank"><i>Materials and Methods.</i></a> The values shown are from a representative experiment out of 3 data sets. They are expressed as a percentage of the activity obtained with control and are the mean (±SD) of triplicate. Statistical analysis performed using the Student t test (**p<0.005 and ***p<0.0001). (<b>E</b>) MCF-7 cells were transiently transfected with the human and murine RIP140 promoter reporter plasmids (25 ng) together with expression vectors for E2F1, DP1 (25 ng each). Results were expressed as described in panel D and a representative experiment is shown (n = 7).