Fig. 2. E2F mobility-shift assays of nuclear extracts from synchronized G₁ and TGF-ß1-induced preapoptotic cells. Nuclear extracts were prepared, incubated with wild-type ³²P-labeled E2F oligonucleotide probe and analyzed by PAGE (lanes 1-12) as described in Materials and Methods. Unlabeled wild-type E2F oligonucleotides were added at 10-fold (lanes 5,9), 100-fold (lanes 6,10) and 1000-fold (lanes 7,11) molar excess, or a 1000-fold molar excess of unlabeled mutant E2F oligonucleotide (lanes 8,12) during the 30 minute incubation for binding specificity. Negative controls containing no nuclear extracts were devoid of activity (lanes 13,14). The position and numerical designation of the ³²P-labeled DNA—protein complexes are indicated on the left. The cell-cycle-associated phases of the nuclear extracts are indicated at the top. mt, mutant E2F oligonucleotide; TGF-ß1, preapoptotic nuclear extract from HuH-7 cells incubated with TGF-ß1 for 72 hours; wt, wild-type E2F oligonucleotide.

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