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. 2003 Mar 15;17(6):723–728. doi: 10.1101/gad.1031803

Figure 5.

Figure 5

Disruption of exogenous and endogenous dE2F2/RBF1 complexes. (A) E2F EMSA assays showing the E2F complexes present in SL2 extracts. Transfection of pIE4-dE2F2 and pIE4-RBF1 generates a dE2F2/RBF1 complex that is disrupted by the coexpression of CycE/cdk2. (B) ChIP assays showing the enrichment of E2F-regulated promoters (PCNA and DNA polα) over a negative control (rp49) in dE2F2 or RBF immunoprecipitates that were prepared from extracts of control-treated cells, asynchronous dE2F1/DAP-depleted cells, or hydroxyurea-treated dE2F1/DAP-depleted cells. RBF1 is no longer detected at these promoters when cells are arrested in S phase with hydroxyurea. (C) Immunoprecipitation (IP)/Western analysis of the cells treated in B showing that RBF1 is present in the whole cell extracts (WCX) but no longer coprecipitates with dE2F2 in hydroxyurea-treated dE2F1- and DAP-depleted cells. RαM is a negative control polyclonal antibody. (D) Expression of CycE/cdk2 increases transcriptional activation by dE2F1 (50 ng pIE4-dE2F1) on the PCNA reporter in the presence of dE2F2 (250 ng pIE4-dE2F2) and RBF2 (4 μg pIE4-RBF2). Transfections were as described in Figure 4.