F EMSA competition of the complex between Myc/Max and the fluorescently-labeled IRD-CACGTG probe with increasing concentrations of an unlabeled CACGTG probe (0.195, 0.390, 0.781, 1.563, 3.125, 6.25, 12.50 and 25 μM). The plot at the bottom shows the mean and standard deviation of the quantification of the shifted Myc/Max bands as a function of unlabeled probe concentration (the experiment was performed in triplicate). HEA/WT Kd ratio: as in E. The apparent Kd values were 387 nM for MycWT/Max and 622 nM for MycHEA/Max. G Same as F, with an unlabeled GGATCC probe as competitor. The apparent Kd values were >25 µM for MycWT/Max and 14.5 µM for MycHEA/Max. H Same as F, with an unlabeled CACGTC probe as competitor. The apparent Kd values were 674 nM for MycWT/Max and 280 nM for MycHEA/Max. Note that that last two lanes of the upper gel were loaded in inverted order, and for this reason were cropped and flipped horizontally in the final layout. Da Data information: Note that the differences in apparent Kd values between panels E and F-H can be explained by the different thresholds required to detect quantifiable complexes in the protein titration experiment (E), as opposed to a loss of signal intensity from an already detectable complex in the presence of unlabeled competitor (F-H): as such, competition experiments provide a better approximation of actual binding affinities. (F-H): The plots show the mean and standard deviation. The experiments were performed in triplicate. Unpaired Student t-test was used to compare IC50 values and expressed as p-values. 