E2 binding enhances the chromatin association of Brd4. (A) SiHa cells were electroporated with an empty vector or an HPV11 WT E2, an HPV11 N294A E2, or an HPV11 R37A/I73A E2 expression vector. Twenty-four hours later, cells were fixed and chromatin immunoprecipitated with nonspecific IgG or anti-Brd4 antibodies. DNA was extracted from immunoprecipitates and input fractions and analyzed by qPCR. Enrichment in immunoprecipitated fractions was calculated as a percentage of the input material. The data represent the average of three independent experiments and the standard deviation. The nonspecific IgG values represent the average of vector, WT, N294A, and R37A/I73A samples. (B) SiHa cells were electroporated with an empty vector or an HPV11 WT E2, an HPV11 N294A E2, or an HPV11 R37A/I73A E2 expression vector. Twenty-four hours later, cells were lysed and E2 was coimmunoprecipitated (IP) with anti-Brd4 antibodies. Immunoprecipitates and input fractions were separated electrophoretically and immunoblotted with primary mouse monoclonal antibodies for HPV11 E2 and an anti-mouse IgG secondary antibody conjugated with horseradish peroxidase. (C) Western blot analysis of E2 levels in the cells used in ChIP assay. Cells (105) were lysed, separated electrophoretically, and immunoblotted with primary mouse monoclonal antibodies for HPV11 E2 and an anti-mouse IgG α-tubulin and secondary antibody conjugated with horseradish peroxidase.