FIG. 6.

Intact LXCXE motif and J domain are required to disrupt endogenous E2F DNA binding complexes. Gel retardation assays were performed with extracts prepared from confluent MEFs that stably expressed either TAg, the LXCXE mutant K1, the J domain mutant H42Q, or the chimeric protein HSJ1-T. (A) Extracts were incubated with an oligonucleotide from the DHFR promoter as in Fig. 5. (B) The complexes observed with extracts from cells expressing K1 or H42Q (lanes 2 and 3 from panel A) were incubated with a panel of antibodies. (C) Immunoprecipitation-DOC release of E2F DNA binding activity. Extracts prepared from confluent MEFs were immunoprecipitated with the control antibody M73 (anti-adenovirus E1A [lanes 1 to 4]) or with anti-p130 antibody (lanes 5 to 8). Immune complexes were denatured and then renatured as described in Materials and Methods and incubated with the DHFR promoter as in Fig. 4.