FIG. 2.

ANCCA interacts with E2Fs and mediates E2F-dependent gene expression. (A) Nuclear extracts prepared from asynchronously growing breast cancer cells were immunoprecipitated with the indicated antibodies. The presence of ANCCA in the precipitates was analyzed by Western blotting with anti-ANCCA antibody. (B, C) Purified, recombinant Flag-tagged ANCCA protein was used in the pulldown experiment with bead-bound GST-E2F proteins (B) or GST-E2F1 fragments (C), which were displayed by Ponceau S staining. ANCCA retained on the beads was detected by Western blotting with anti-Flag antibody. Amino acid number and domains contained in the fragments of human E2F1 are indicated. CAD, cyclin A binding domain; DBD, DNA binding domain; DIM, dimerization domain; TA, transactivation domain; and PB, pocket protein binding domain. Arrows in panel B indicate the full lengths of different E2F-GST fusion proteins. (D) Recombinant Flag-tagged E2F1 protein was used in pulldown experiments with bead-bound GST-ANCCA fragments. E2F1 protein retained on glutathione beads was detected by immunoblotting with anti-Flag antibody. Loading of GST proteins was visualized by Ponceau S staining. D/E, aspartate- and glutamate-rich region; AAA D1 or D2, AAA⁺ domains; Bromo, bromodomain. (E) HeLa cells were transfected with a luciferase reporter construct containing the human cyclin E1 promoter or synthetic promoter bearing 3 copies of E2F binding sites. Cells were also cotransfected with E2F1- and/or ANCCA-expressing plasmids as indicated. Luciferase activity data are the means of results from three independent experiments. (F) Cells were cotransfected with the reporters, ANCCA or control siRNA, E2F1, and/or siRNA-protected ANCCA-expressing (ANCCA*) plasmids as indicated. Fold induction was obtained by comparison of normalized luciferase activities from different cells with the one transfected only with control siRNA set as 1. Data are representative of results from three independent experiments. (G) HeLa cells were cotransfected with ANCCA or control siRNA and GFP or siRNA-protected ANCCA expression plasmid (ANCCA*). Two days after transfection, cells were harvested for Western analysis with the indicated antibodies.