Dimerization and DNA binding properties of E2F-6 (A) C33-A cells were transiently transfected with expression vectors encoding E2F-1, E2F-4, or HA–E2F-6 in the presence or absence of DP (DP-1 or DP-2) or pocket proteins (pRB, p107) and immunoprecipitated with the indicated antibodies. (B) C33-A cells were transiently transfected with the identical combinations of expression vectors as in A. Gel shift assays were carried out on the indicated unlabeled cell extracts (1.5 μg of total protein per lane) by using the consensus E2F site from the adenoviral E2 promoter (TTTCGCGCCCTTT). Western blot assays were carried out on the same unlabeled cell extracts (300 ng of total protein per lane) by using monoclonal antibodies against E2F-1 (KH95), E2F-4 (LLF4–1), or the HA tag (12CA5). Gel retardation assays have also been conducted by using the additional E2F sites TTTCCCGCCTTT, TTTCCCGCCAAA, TTTCCCGCGTGT, or ATTCCCGCGCTTT with similar differences in affinity.