Figure 6.

Interaction between TopBP1 and E2F1 during G1/S transition. (A) Primary human foreskin fibroblasts (HFF) were brought to quiescence by serum starvation for 48 h and then stimulated by 20% FBS for indicated periods of time. The cells were fixed and analyzed with propidium iodide staining by flow cytometry. (B) Cell lysates from synchronized HFF were subjected to Western blot analysis, and the immunoblot was probed with antibodies specific to TopBP1, E2F1, and β-actin, respectively. (Bottom panel) The fractions of each phase of cell cycle quantitated by FACS analysis. (C) The endogenous E2F1 in the whole-cell lysates of synchronized HFF was immunoprecipitated with an anti-E2F1 antibody (KH95) or control mouse IgG and resolved by SDS-PAGE; the immunoblotting was performed as indicated. (Lower panel) An aliquot of lysates was subjected to Western blot analysis. The immunoblot was probed with a TopBP1 antibody. (D) TopBP1 is an E2F target. HFF cells were serum-starved for 48 h and then infected with an adenovirus expressing HA-E2F1 (AdE2F1) or that harboring a CMV empty vector (AdCMV) at a multiplicity of infection of 400 and 1000. Some AdCMV-infected cells were stimulated with 20% serum, and the others remained in 0.1% serum. The cells were harvested at 21 h after infection. (Left panel) The expression of TopBP1 and E2F1 in the whole-cell lysates was detected with their specific antibodies. (Right panel) Poly(A) RNA was prepared from infected HFF, and Northern blot analysis was carried out with a TopBP1 cDNA probe or GAPDH probe.