Fig. 8. Gel retardation analysis of complex formation on the E2F-binding site. Reh cells were treated with forskolin (100 µM) for the hours indicated. (A) Total extracts were prepared as described in Materials and Methods and incubated with ³²P-labelled oligonucleotide probe containing the E2F-consensus binding site. (B,C) Supershift assays were performed by incubating total cell extract with the appropriate antibodies before adding the probe containing the E2F-binding site. In all three panels, the protein-DNA complexes were subjected to a native 4% polyacrylamid gel electrophoresis and visualised by autoradiography. One representative experiment of three is shown. I, II and III denote the different protein complexes bound to the E2F-probe. The asterisks indicate supershifted bands.