Figure 5.

JAZ inhibits E2F1's specific DNA binding in vitro. (A) HA-E2F1/pcDNA3 (pcDNA3 as a control) was transfected alone or co-transfected with FLAG-JAZ/pcDNA3 into NIH3T3 cells (the amount of DNA was kept constant using pcDNA3). After 24 h, cells were harvested, and nuclear extracts were prepared and subjected to EMSA analysis with a ³²P-labeled DNA probe that contained a wild-type E2F binding site. (B) An EMSA analysis was also performed using Raji nuclear extract containing endogenous E2F1 with the ³²P-labeled E2F probe in the presence or absence of an E2F1 antibody [for induction of supershift(s)] as described in Materials and Methods. Purified GST-JAZ (GST as a control) or His-JAZ recombinant protein was added in the EMSA assay to assess the effect of JAZ on E2F1's DNA binding. Excessive amounts of unlabeled wild-type or mutant E2F oligonucleotides were also added in the controls to verify the binding specificity.