U2OS cells (HTB-96, American Type Culture Collection; RRID: CVCL_0042) were plated on coverslips and transfected for 48 hours with the indicated plasmids, or U2OS-Tet-ON cells were induced to express WT E2F1, E2F1-KK, or E2F1-R109K for 24 hours as appropriate. Cells were fixed for 15 min with 4% paraformaldehyde in PBS and permeabilized for 15 min with 0.5% Triton X-100 in PBS. Coverslips were incubated with primary antibody for 1 hour, washed five times, and then incubated with Alexa Fluor 488–conjugated secondary antibody (Thermo Fisher Scientific; RRID: AB_141607) for 1 hour. Coverslips were washed again before mounting on glass slides using VECTASHIELD mounting medium with DAPI (4′,6-diamidino-2-phenylindole; Vectorlabs). Proteins were visualized on a BX60 fluorescence microscope (Olympus) fitted with a Hamamatsu C4742-95 camera and analyzed with Openlab 5 software (Improvision).

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