Figure 4. Autoradiograms of the EMSA experiments.

(A) Competition experiments with the proximal CCAAT box of the histone H3/k promoter (KProx). Composition of the individual incubation mixtures: labelled oligonucleotides are marked with an *, proteins added (HNE, HeLa nuclear extract; NF-Y, isolated NF-Y) are indicated by ±, competitors (80-fold molar excess) and antibody are indicated in the lower part of the heading. The NF-Y double bands are marked by black arrowheads. It may be noted that the specific activity of the NF-Y * probe used was much lower than the specific activity of the KProx* probe. (B) Supershift and complex-binding experiments with HNE and recombinant NF-Y. The experimental set-up and the heading is the same as for (A). To the incubation mixture indicated by ab CBF-A, 1 μg of antibody raised against NF-YB was added. Approx. 100 ng of isolated NF-Y complex was added to the labelled oligonucletides where indicated. The supershift band is indicated by the open arrow. (C) Titration competition experiments. The binding of labelled double-stranded oligonucleotides containing the consensus sequence of the binding site of NF-Y was competed by increasing concentrations of unlabelled double-stranded oligonucleotides (NF-Y, KProx and KDist). The molar excess of unlabelled competitor oligonucleotide is indicated. The intensities of the signals were quantified with respect to the PhosphoImager signals by the Aida 2.2 software from Raytest and expressed as the percentage ratio to the uncompeted signal intensities.