Figure 4. APC/CCdh1 targets E2F7 and E2F8 for degradation. (A) Normalized expression levels of E2F1, E2F7, E2F8, and cyclin A2 (CCNA2) in the depicted B-cell types, according to GEO database GSE4142. (B) The proteins XlTome-1, hE2F7, and hE2F8 were expressed in rabbit reticulocytes supplemented with ³⁵S-methionine, and incubated in G₁ phase S3 cell extracts supplemented with wt or dominant negative (DN) Ubch10 and either buffer, MG132, the C-terminus of Emi1, or unlabeled Securin. Time-dependent degradation was assayed by SDS-PAGE and autoradiography (top), and quantified (bottom). (C) HEK293 cells were co-transfected with Cdh1 or empty vector (pCS2) and with either E2F8, or E2F7-EGFP, or EGFP, at a 4:1 ratio. After 30 h, cells were harvested for western blotting with E2F8, GFP (experimental control), and tubulin (loading control) antibodies. (B and C) are representative experiments. (D and E) Model for the interplay between APC/CCdh1 and E2F1, E2F7, and E2F8. APC/CCdh1 regulates the proteolysis of both E2F1 and its repressors, E2F7 and E2F8 (B and C) during G₁-phase. The model proposes that a delicate difference in the timing of proteolysis of the activating vs. repressive E2F transcription factors controls the G₁/S transition of the mammalian cell cycle (see main text for further information/discussion).