Determination of comparative dissociation rate of NFY when interacting with consensus and nonconsensus binding sequences.

HeLa cell nuclear extracts (8 μg) and oligonucleotide probes R and IR (corresponding to sequences +215 to +247 and −30 to +1, respectively) were used in gel mobility experiments as previously described. (A) For the concentration-dependent dissociation rate experiment, DNA probes and nuclear extracts were incubated for 20 minutes in the presence of various concentrations of double-stranded NFY competitor (ranging from 1×-100× excess of probe). (B) For the time-dependent dissociation rate experiment, the oligonucleotide probes were incubated independently with nuclear extract for 10 minutes prior to the addition of a 50-fold excess of double-stranded NFY competitor. Reaction mixtures were further incubated at 4 degrees for various time points (minutes) as shown above each lane. Complexes formed in both time- and concentration-dependent experiments (A and B) were analyzed on 5% nondenaturing acrylamide gel as described in Figure 2. Lane “−” in both A and B represents the DNA-NFY protein complex formed in the absence of competitor. (C, D) Densities of protein-DNA complexes were quantitated by phosphorimager analysis and the value obtained for protein-DNA complexes in the absence of competitors were used as control references. Densities of all other protein-DNA complexes in the presence of competitors were determined as percentages of control references for each experiment. The graphs represent the average of 2 independent analyses for each authoradiograph and the error bars represent the standard deviations. The solid circle represents the values obtained for the protein complex with the IR (−30 to +1) probe, and the solid triangle represents the values obtained for the protein complex with the R (+215 to +247) probe.